Processes for producing fermentation products

ABSTRACT

The present invention relates to processes for producing fermentation products from starch-containing material, wherein a bacterial alpha-amylase, a raw starch hydrolyzing alpha-amylase and a carbohydrate-source generating enzyme are present and/or added during liquefaction. The invention also relates to compositions suitable for use in processes of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 14/351,471 filed Apr. 11, 2014, now U.S. Pat. No. 9,677,095, which is a 35 U.S.C. 371 national application of PCT/US2012/059335 filed Oct. 9, 2012, which claims priority or the benefit under 35 U.S.C. 119 U.S. provisional application Nos. 61/545,865 and 61/566,165, filed Oct. 11, 2011 and Dec. 2, 2011, respectively, the contents of which are fully incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to processes for producing fermentation products from starch-containing material. The invention also relates to compositions suitable for use in processes of the invention.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Production of fermentation products, such as ethanol, from starch-containing material is well-known in the art. Industrially two different kinds of processes are used today. The most commonly used process, often referred to as a “conventional process”, including liquefying gelatinized starch at high temperature (typically between 80-90° C. at a pH between 5 and 6) using a bacterial alpha-amylase, followed by simultaneous saccharification and fermentation (SSF) carried out in the presence of a glucoamylase and a fermentation organism. Another well known process, often referred to as a “raw starch hydrolysis”-process (RSH process) includes simultaneously saccharifying and fermenting granular starch below the initial gelatinization temperature typically in the presence of an acid fungal alpha-amylase and a glucoamylase.

Despite significant improvements of fermentation product production processes over the past decade a significant amount of residual starch material is not converted into the desired fermentation product, such as ethanol. Therefore, there is still a desire and need for providing processes for producing fermentation products, such as ethanol, from starch-containing material that can provide a higher fermentation product yield compared to a conventional process.

SUMMARY OF THE INVENTION

The present invention relates to processes of producing fermentation products, such as especially ethanol, from starch-containing material using a fermenting organism.

In the first aspect the invention relates to processes for producing a fermentation product, such as ethanol, comprising the steps of:

i) liquefying a starch-containing material at a temperature in the range from 60-80° C. using:

-   -   a bacterial alpha-amylase;     -   a raw starch hydrolyzing alpha-amylase;     -   a carbohydrate-source generating enzyme having a heat stability         at 70° C., pH 5.3, of at least 70%;

ii) saccharifying using a carbohydrate-source generating enzyme;

iii) fermenting using a fermenting organism.

In an embodiment also a protease, such as a metallo protease, is present and/or added during liquefaction in step i).

In an embodiment a pullulanase also is present and/or added during liquefaction in step i).

In an embodiment the bacterial alpha-amylase is derived from a strain of the genus Bacillus, preferably a strain of Bacillus stearothermophilus. The Bacillus stearothermophilus alpha-amylase may be truncated. In an embodiment the bacterial alpha-amylase is derived from Bacillus stearothermophilus (SEQ ID NO: 3 in WO 99/019467 or SEQ ID NO: 1 herein) which may be truncated to have about 491 amino acids. In an embodiment the Bacillus stearothermophilus alpha-amylase is truncated as indicated above and further has I181*+G182* deletions (relative to SEQ ID NO: 3 in WO 99/019467 or SEQ ID NO: 1 herein) or I181*+G182* deletions and a N193F substitution. In a preferred embodiment the bacterial alpha-amylase is derived from Bacillus stearothermophilus alpha-amylase (SEQ ID NO: 3 in WO 99/019467 or SEQ ID NO: 1 herein) which truncated, e.g., to have about 491 amino acids. The truncated Bacillus stearothermophilus alpha-amylase may also have mutations selected from the group consisting of:

-   -   V59A+Q89R+E129V+K177L+R179E+I181*+G182*+N193F+H208Y+K220P+N224L+Q254S;     -   E129V+K177L+R179E+I181*+G182*+N193F; and     -   E129V+K177L+R179E+I181*+G182*+N193F+K220P+N224L+S242Q+Q254S.

In an embodiment the raw starch hydrolyzing alpha-amylase is of fungal origin. In a preferred embodiment the raw starch hydrolyzing alpha-amylase is derived from Rhizomucor pusillus with Aspergillus niger glucoamylase linker and SBD. In a specific embodiment the raw starch hydrolyzing alpha-amylase is a variant of Rhizomucor pusillus alpha-amylase with Aspergillus niger glucoamylase linker and SBD with further one or more of the following substitutions: G128D, D143N, K192R, such as G128D+D143N or G128D+D143N+K192R (using SEQ ID NO: 14 herein for the numbering).

In another embodiment the raw starch hydrolyzing alpha-amylase is derived from Aspergillus, such as Aspergillus niger alpha-amylase with Aspergillus kawachii linker and SBD or the Aspergillus kawachii alpha-amylase itself.

In an embodiment the carbohydrate-source generating enzyme present and/or added during liquefaction is different from the carbohydrate-source generating enzyme present and/or added during saccharification and/or fermentation.

Especially contemplated carbohydrate-source generating enzymes are glucoamylases. In a preferred embodiment the glucoamylase added during liquefaction is from the genus Penicillium, especially a strain of Penicillium oxalicum disclosed as SEQ ID NO: 2 in PCT/CN10/071753 published as WO 2011/127802 (which is hereby incorporated by reference) and shown in SEQ ID NO: 9 or 15 herein, or a protease stable protein engineered variant of the Penicillium oxalicum glucoamylase disclosed in U.S. application No. 61/531,189 or U.S. application No. 61/566,046 or PCT/US12/053779 having a K79V substitution.

In a preferred embodiment the carbohydrate-source generating enzyme is a variant of the Penicillium oxalicum glucoamylase disclosed as SEQ ID NO: 2 in PCT/CN10/071753 published as WO 2011/127802 and shown in SEQ ID NO: 9 and 15 herein, having a K79V substitution (using the mature sequence shown in SEQ ID NO: 15 for numbering). The K79V glucoamylase variant has reduced sensitivity to protease degradation relative to the parent as disclosed in U.S. application No. 61/531,189 and U.S. application No. 61/566,046 or PCT/US12/053779 (which are hereby incorporated by reference).

In a preferred embodiment the carbohydrate-source generating enzyme present and/or added during saccharification and/or fermentation, such as SSF, is a glucoamylase of fungal origin, preferably from a strain of Aspergillus, preferably Aspergillus niger, Aspergillus awamori, or Aspergillus oryzae; or a strain of Trichoderma, preferably T. reesei; or a strain of Talaromyces, preferably T. emersonii. In an embodiment the glucoamylase present and/or added during saccharification and/or fermentation may be derived from a strain of the genus Pycnoporus, in particular a strain of Pycnoporus as described in WO 2011/066576 (Novozymes), or from a strain of the genus Gloephyllum, in particular a strain of Gloephyllum as described in WO 2011/068803 (Novozymes) or a strain of the genus Nigrofomes, in particular a strain of Nigrofomes sp. disclosed in PCT/US10/058375 published as WO 2012/064351 (Novozymes).

In a second aspect the invention relates to a composition comprising

-   -   a bacterial alpha-amylase;     -   a raw starch hydrolyzing alpha-amylase;     -   a carbohydrate-source generating enzyme having a heat stability         at 70° C., pH 5.3, of at least 70%.

In an embodiment of the invention a protease, such as a metallo protease, and/or a pullulanase is included in the composition. Other enzymes may also be included.

Examples of suitable bacterial alpha-amylases, raw starch hydrolyzing alpha-amylases and carbohydrate-source generating enzymes, especially glucoamylases, can be found below in the “Enzymes”-section.

BRIEF DESCRIPTION OF THE FIGURES

The FIGURE shows the ethanol concentration after liquefaction at 75° C., pH 4.8, with Bacillus stearothermophilus alpha-amylase variant, raw starch hydrolyzing alpha-amylase variant derived from Rhizomucor pusillus, and Penicillium oxalicum glucoamylase variant followed by SSF for 54 hours using a Talaromyces emersonii glucoamylase and Saccharomyces cerevisae yeast.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to processes of producing fermentation products, such as especially ethanol, from starch-containing material using a fermenting organism.

The inventors have shown that a process of the invention has a number of advantages. Example 5 shows that the yield is increased in an ethanol process where a combination of bacterial alpha-amylase, raw starch degrading alpha-amylase, and glucoamylase is present during liquefaction at 75° C., pH 4.8, followed by 54 hours simultaneous saccharification and fermentation (SSF) with Saccharomyces cerevisae yeast.

In the first aspect the invention relates to processes for producing fermentation products, such as ethanol, from starch-containing material comprising the steps of:

i) liquefying the starch-containing material at a temperature in the range from 60-80° C. using:

-   -   a bacterial alpha-amylase;     -   a raw starch hydrolyzing alpha-amylase;     -   a carbohydrate-source generating enzyme having a heat stability         at 70° C., pH 5.3, of at least 70%;

ii) saccharifying using a carbohydrate-source generating enzyme;

iii) fermenting using a fermenting organism.

In an embodiment the process of the invention further comprises, prior to the liquefaction step i), the steps of:

a) reducing the particle size of the starch-containing material, preferably by dry milling;

b) forming a slurry comprising the starch-containing material and water.

The slurry may contain from 10-55 w/w-% dry solids (DS), preferably 25-45 w/w-% dry solids (DS), more preferably 30-40 w/w-% dry solids (DS) of starch-containing material.

The starch-containing material used as starting material is typically reduced in particle size, e.g., by milling, in order to open up the structure and allowing for further processing.

Generally there are two types of milling: wet and dry milling. In dry milling whole kernels are milled and used. Wet milling gives a good separation of germ and meal (starch granules and protein) and is often applied at locations where the starch hydrolysate is used in production of, e.g., syrups. Both dry and wet milling is well known in the art. According to the invention dry milling is preferred. In an embodiment the particle size is reduced to between 0.05 to 3.0 mm, preferably 0.1-0.5 mm, or so that at least 30%, preferably at least 50%, more preferably at least 70%, even more preferably at least 90% of the starch-containing material fit through a sieve with a 0.05 to 3.0 mm screen, preferably 0.1-0.5 mm screen. In another embodiment at least 50%, preferably at least 70%, more preferably at least 80%, especially at least 90% of the starch-containing material fit through a sieve with #6 screen.

The pH during liquefaction step i) is typically in the range from 4-6, preferably from 4.5-5.0 or 4.5-4.8 or between 5 and 6. The temperature during liquefaction may be in the range between from 70-80° C., such as between 75-80° C., preferably around 75° C. Typically the starch-containing material is heated during liquefaction step i) for 0.1-10 hours, such as 1-3 hours, such as around 1.5 hours.

The bacterial alpha-amylase, raw starch hydrolyzing alpha-amylase, and carbohydrate-source generating enzyme, in particular glucoamylase, and optional protease and/or pullulanase may be added to the aqueous slurry to initiate liquefaction (thinning). In an embodiment a part of the enzyme blend is added to the aqueous slurry, while the rest of the enzyme is added during liquefaction step i).

The aqueous slurry may in an embodiment be jet-cooked to further gelatinize the slurry before being subjected to liquefaction in step i). The jet-cooking may be carried out at a temperature between 95-145° C., such as 105-125° C., e.g., 110-145° C., preferably 120-140° C., such as 125-135° C., preferably around 130° C. for about 1-15 minutes, preferably for about 3-10 minutes, especially around about 5 minutes.

In an embodiment saccharification steps ii) and fermentation step iii) are carried out either sequentially or simultaneously. In a preferred embodiment steps ii) and iii) are carried out simultaneously (SSF process). In a preferred embodiment a carbohydrate-source generating enzyme, preferably a glucoamylase, is added. The carbohydrate-source generating enzyme, such as a glucoamylase, may be different from the enzyme added during liquefaction step i).

In an embodiment saccharification step ii) is carried out at a temperature from 20-75° C., preferably from 40-70° C., such as around 60° C., and at a pH between 4 and 5, such as around pH 4.5.

Further, fermentation step iii) or simultaneous saccharification and fermentation (SSF) may be carried out at a temperature from 25-40° C., such as from 28-35° C., such as from 30-34° C., preferably around about 32° C., wherein fermentation is ongoing for 6-120 hours, in particular 24-96 hours, such as around 54 hours.

Examples of suitable bacterial alpha-amylases can be found in the “Bacterial Alpha-Amylases” section below. In a preferred embodiment the bacterial alpha-amylase is a Bacillus alpha-amylase, preferably derived from a strain of Bacillus stearothermophilus, in particular the one shown in SEQ ID NO: 3 in WO 99/019467 or SEQ ID NO: 1 herein, such as one that is truncated, e.g., to have around 491 amino acids, e.g., from 485-495 amino acids. The Bacillus stearothermophilus alpha-amylase may also be a variant, e.g., one of the ones listed below and/or disclosed in WO 2011/082425 (hereby incorporated by reference).

Examples of raw starch hydrolyzing alpha-amylases can be found in the “Raw Starch Hydrolyzing Alpha-Amylases” section below. In an embodiment the raw starch hydrolyzing alpha-amylase is of fungal origin. In a preferred embodiment the raw starch hydrolyzing alpha-amylase is derived from Rhizomucor pusillus alpha-amylase and has an Aspergillus niger glucoamylase linker and SBD. In a preferred embodiment the raw starch hydrolyzing alpha-amylase is a variant of above further having one of the following substitutions: G128D+D143N or G128D+D143N+K192R (using SEQ ID NO: 14 herein for the numbering).

Examples of carbohydrate-source generating enzymes, including in particular glucoamylases, and can be found in the “Carbohydrate-Source Generating Enzymes” section below. The carbohydrate-source generating enzyme has a heat stability at 70° C., pH 5.3, of at least 70%, such as at least 75%, preferably at least 80%, preferably at least 85%.

In a preferred embodiment the glucoamylase is from the genus Penicillium, especially a strain of Penicillium oxalicum disclosed as SEQ ID NO: 2 in PCT/CN10/071753 published as WO 2011/127802 (which is hereby incorporated by reference) and shown in SEQ ID NO: 9 and 15 herein or a protease stable protein engineered variant of the Penicillium oxalicum glucoamylase disclosed in U.S. application No. 61/531,189 or U.S. application No. 61/566,046 or PCT/US12/053779 having a K79V substitution.

In an embodiment a protease is also present during liquefaction step i). Examples of proteases can be found in the “Proteases”-section below. In an embodiment the protease is a metallo protease. In an preferred embodiment the protease is derived from the genus Thermoascus, preferably a strain of Thermoascus aurantiacus, especially Thermoascus aurantiacus CGMCC No. 0670 (classified as EC 3.4.24.39) disclosed in SEQ ID NO: 3 herein or amino acids 1 to 177 (the mature polypeptide) of SEQ ID NO: 1 of WO 2010/008841.

Starch-Containing Materials

Any suitable starch-containing material may be used according to the present invention. The starting material is generally selected based on the desired fermentation product. Examples of starch-containing materials, suitable for use in processes of the invention, include whole grains, corn, wheat, barley, rye, milo, sago, cassava, tapioca, sorghum, rice, peas, beans, or sweet potatoes, or mixtures thereof or starches derived there from, or cereals. Contemplated are also waxy and non-waxy types of corn and barley. Preferred starch-containing materials are corn and wheat.

Saccharification and Fermentation

One or more carbohydrate-source generating enzymes, in particular glucoamylases, are present and/or added during saccharification step ii) and/or fermentation step iii). The carbohydrate-source generating enzyme may preferably be a glucoamylase, but may also be an enzyme selected from the group consisting of: beta-amylase, maltogenic amylase and alpha-glucosidase.

Examples of carbohydrate-source generating enzymes, including glucoamylases, can be found in the “Carbohydrate-Source Generating Enzyme Present and/or Added During Saccharification and/or Fermentation”-section below.

When doing sequential saccharification and fermentation the saccharification in step ii) may be carried out using conditions well-known in the art. For instance, the saccharification step ii) may last up to from about 24 to about 72 hours. However, it is common to do only a pre-saccharificaton of typically 40-90 minutes at a temperature between 30-65° C., typically about 60° C., followed by saccharification during fermentation in simultaneous saccharification and fermentation (SSF). Saccharification may be carried out at temperatures from 20-75° C., preferably from 40-70° C., typically around 60° C., and at a pH between 4 and 5, normally at about pH 4.5.

Simultaneous saccharification and fermentation (SSF) is widely used in industrial scale fermentation product production processes, especially ethanol production processes. When doing SSF the saccharification step ii) and the fermentation step iii) are carried out simultaneously. There is no holding stage for the saccharification, meaning that a fermenting organism, such as yeast, and enzyme(s), may be added together. SSF is according to the invention typically carried out at a temperature from 25° C. to 40° C., such as from 28° C. to 35° C., such as from 30° C. to 34° C., preferably around about 32° C. In an embodiment fermentation is ongoing for 6 to 120 hours, in particular 24 to 96 hours. In an embodiment the pH is between 3.5-5, in particular between 3.8 and 4.3.

Fermentation Medium

“Fermentation media” or “fermentation medium” which refers to the environment in which fermentation is carried out and which includes the fermentation substrate, that is, the carbohydrate source that is metabolized by the fermenting organism. The fermentation medium may comprise nutrients and growth stimulator(s) for the fermenting organism(s). Nutrient and growth stimulators are widely used in the art of fermentation and include nitrogen sources, such as ammonia; urea, vitamins and minerals, or combinations thereof.

Fermenting Organisms

The term “Fermenting organism” refers to any organism, including bacterial and fungal organisms, suitable for use for fermentation in a process of the invention. The fermenting organism is capable of producing the desired fermentation product. Especially suitable fermenting organisms are able to ferment, i.e., convert, sugars, such as glucose or maltose, directly or indirectly into the desired fermentation product, such as ethanol. Examples of fermenting organisms include fungal organisms, such as yeast. Preferred yeast, especially for ethanol production, includes strains of Saccharomyces spp., in particular, Saccharomyces cerevisiae.

In one embodiment the fermenting organism is added to the fermentation medium so that the viable fermenting organism, such as yeast, count per mL of fermentation medium is in the range from 10⁵ to 10¹², preferably from 10⁷ to 10¹⁰, especially about 5×10⁷.

Commercially available yeast includes, e.g., RED STAR ETHANOL RED™ yeast (available from Fermentis/Lesaffre, USA), FALI (available from Fleischmann's Yeast, USA), SUPERSTART and THERMOSACC™ fresh yeast (available from Ethanol Technology, WI, USA), BIOFERM AFT and XR (available from NABC—North American Bioproducts Corporation, GA, USA), GERT STRAND (available from Gert Strand AB, Sweden), and FERMIOL (available from DSM Specialties).

Fermentation Products

The term “fermentation product” means a product produced by a process including a fermentation step using a fermenting organism. Fermentation products contemplated according to the invention include alcohols (e.g., ethanol, methanol, butanol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, succinic acid, gluconic acid); ketones (e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H₂ and CO₂); antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins (e.g., riboflavin, B₁₂, beta-carotene); and hormones. In a preferred embodiment the fermentation product is ethanol, e.g., fuel ethanol; drinking ethanol, i.e., potable neutral spirits; or industrial ethanol or products used in the consumable alcohol industry (e.g., beer and wine), dairy industry (e.g., fermented dairy products), leather industry and tobacco industry. Preferred beer types comprise ales, stouts, porters, lagers, bitters, malt liquors, happoushu, high-alcohol beer, low-alcohol beer, low-calorie beer or light beer. Preferred fermentation processes used include alcohol fermentation processes. The fermentation product is preferably ethanol. The fermentation products obtained according to the process of the invention may be ethanol. The fermentation product, such as especially ethanol, may be used as fuel which is typically blended with gasoline. However, in the case of ethanol it may also be used as potable ethanol. In a preferred embodiment the fermentation product is fuel ethanol.

Recovery

Subsequent to fermentation the fermentation product may be separated from the fermentation medium. The slurry may be distilled to extract the desired fermentation product. Alternatively the desired fermentation product may be extracted from the fermentation medium by micro or membrane filtration techniques. The fermentation product may also be recovered by stripping or other method well known in the art.

Enzymes

Bacterial Alpha-Amylases

According to the invention a bacterial alpha-amylase is present and/or added during liquefaction together of a raw starch hydrolyzing enzyme and a carbohydrate-source generating enzyme having a heat stability at 70° C., pH 5.3, of at least 70%, such as at least 75%, preferably at least 80%, preferably at least 85%.

Optionally a protease and/or a pullulanase is present or added during liquefaction as well.

The term “bacterial alpha-amylase” means any bacterial alpha-amylase classified under EC 3.2.1.1. A bacterial alpha-amylase used according to the invention may, e.g., be derived from a strain of the genus Bacillus, which is sometimes also referred to as the genus Geobacillus. In a preferred embodiment the Bacillus alpha-amylase is derived from a strain of Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus stearothermophilus, or Bacillus subtilis, but may also be derived from other Bacillus sp.

Specific examples of bacterial alpha-amylases include the Bacillus amyloliquefaciens alpha-amylase of SEQ ID NO: 5 in WO 99/19467, the Bacillus licheniformis alpha-amylase of SEQ ID NO: 4 in WO 99/19467, and the Bacillus stearothermophilus alpha-amylase of SEQ ID NO: 3 in WO 99/19467 (all sequences are hereby incorporated by reference). In an embodiment the alpha-amylase may be an enzyme having a degree of identity of at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% to any of the sequences shown in SEQ ID NO: 3, 4 or 5, respectively, in WO 99/19467.

In an embodiment the bacterial alpha-amylase may be an enzyme having a degree of identity of at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% or at least 100% to any of the sequences shown in SEQ ID NO: 3 in WO 99/19467 or SEQ ID NO: 1 herein.

The Bacillus alpha-amylase may also be a variant and/or hybrid. Examples of such a variant can be found in any of WO 96/23873, WO 96/23874, WO 97/41213, WO 99/19467, WO 00/60059, and WO 02/10355 (all documents are hereby incorporated by reference). Specific alpha-amylase variants are disclosed in U.S. Pat. Nos. 6,093,562, 6,187,576, 6,297,038, and 7,713,723 (hereby incorporated by reference) and include Bacillus stearothermophilus alpha-amylase (often referred to as BSG alpha-amylase) variants having a deletion of one or two amino acids at positions R179, G180, I181 and/or G182, preferably a double deletion disclosed in WO 96/23873—see, e.g., page 20, lines 1-10 (hereby incorporated by reference), preferably corresponding to deletion of positions 181 and 182 compared to the amino acid sequence of Bacillus stearothermophilus alpha-amylase set forth in SEQ ID NO: 3 disclosed in WO 99/19467 or SEQ ID NO: 1 herein or the deletion of amino acids R179 and G180 using SEQ ID NO: 3 in WO 99/19467 or SEQ ID NO: 1 herein for numbering (which reference is hereby incorporated by reference). Even more preferred are Bacillus alpha-amylases, especially Bacillus stearothermophilus alpha-amylases, which have a double deletion corresponding to a deletion of positions 181 and 182 and further comprise a N193F substitution (also denoted I181*+G182*+N193F) compared to the wild-type BSG alpha-amylase amino acid sequence set forth in SEQ ID NO: 3 disclosed in WO 99/19467 or SEQ ID NO: 1 herein. The bacterial alpha-amylase may also have a substitution in a position corresponding to S239 in the Bacillus licheniformis alpha-amylase shown in SEQ ID NO: 4 in WO 99/19467, or a S242 variant of the Bacillus stearothermophilus alpha-amylase of SEQ ID NO: 3 in WO 99/19467 or SEQ ID NO: 1 herein. In a preferred embodiment the variant is a S242A, E or Q variant, preferably a S242Q variant of the Bacillus stearothermophilus alpha-amylase (using SEQ ID NO: 1 for numbering).

In an embodiment the variant has an E188 mutation, such as an E188P substitution in the Bacillus stearothermophilus alpha-amylase (using SEQ ID NO: 1 for numbering).

The bacterial alpha-amylase may in a preferred embodiment be a truncated Bacillus licheniformis alpha-amylase. Especially the truncation is so that the Bacillus stearothermophilus alpha-amylase shown in SEQ ID NO: 3 in WO 99/19467 or SEQ ID NO: 1 herein, is around 491 amino acids long.

Bacterial Hybrid Alpha-Amylases

The bacterial alpha-amylase may also be a hybrid bacterial alpha-amylase, e.g., an alpha-amylase comprising 445 C-terminal amino acid residues of the Bacillus licheniformis alpha-amylase (shown in SEQ ID NO: 4 of WO 99/19467) and the 37 N-terminal amino acid residues of the alpha-amylase derived from Bacillus amyloliquefaciens (shown in SEQ ID NO: 5 of WO 99/19467). In a preferred embodiment this hybrid has one or more, especially all, of the following substitutions:

G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S (using the Bacillus licheniformis numbering in SEQ ID NO: 4 of WO 99/19467). Also preferred are variants having one or more of the following mutations (or corresponding mutations in other Bacillus alpha-amylases): H154Y, A181T, N190F, A209V and Q264S and/or the deletion of two residues between positions 176 and 179, preferably the deletion of E178 and G179 (using SEQ ID NO: 5 of WO 99/19467 for position numbering).

In a preferred embodiment the bacterial alpha-amylase is the mature part of the chimeric alpha-amylase disclosed in Richardson et al., 2002, The Journal of Biological Chemistry 277(29): 26501-26507, referred to as BD5088 or a variant thereof. This alpha-amylase is the same as the one shown in SEQ ID NO: 2 in WO 2007/134207. The mature enzyme sequence starts after the initial “Met” amino acid in position 1.

In an embodiment the bacterial alpha-amylase is a thermostable bacterial alpha-amylase. In an embodiment the thermostable bacterial alpha-amylase is one disclosed in WO 2011/082425 (hereby incorporated by reference). In an embodiment the thermostable bacterial alpha-amylase is derived from a strain of the genus Bacillus (or Geobacillus), especially a strain of Bacillus stearothermophilus, in particular the Bacillus stearothermophilus as disclosed in WO 99/019467 as SEQ ID NO: 3 or SEQ ID NO: 1 herein, with the double deletion I181+G182 and substitution N193F, further comprising further mutations:

-   -   V59A+Q89R+G108A+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S+M284V;     -   V59A+Q89R+G112D+E129V+K177L+R179E+K220P+N224L+Q254S;     -   V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;     -   V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S+M284V;     -   V59A+Q89R+E129V+K177L+R179E+K220P+N224L+Q254S+D269E+D281N;     -   V59A+Q89R+E129V+K177L+R179E+K220P+N224L+Q254S+I270L;     -   V59A+Q89R+E129V+K177L+R179E+K220P+N224L+Q254S+H274K;     -   V59A+Q89R+E129V+K177L+R179E+K220P+N224L+Q254S+Y276F;     -   V59A+G108A;     -   V59A+G108A+E129V+K177L+R179E+H208Y+K220P+N224L+S242Q+Q254S+M284V;     -   V59A+G108A+S242Q+M284V;     -   V59A+G108A+M284V;     -   V59A+E129V+R157Y+K177L+R179E+K220P+N224L+S242Q+Q254S;     -   V59A+E129V+K177L+R179E+H208Y+K220P+N224L+S242Q+Q254S;     -   V59A+E129V+K177L+R179E+H208Y+K220P+N224L+S242Q+Q254S+M284V;     -   V59A+E129V+K177L+R179E+H208Y+M284V;     -   V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S;     -   V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+H274K;     -   V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+Y276F;     -   V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+D281N;     -   V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+M284T;     -   V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+G416V;     -   V59A+E129V+K177L+R179E+K220P+N224L+Q254S;     -   V59A+E129V+K177L+R179E+K220P+N224L+Q254S+M284T;     -   V59A+H208Y+K220P+N224L+Q254S+M284V;     -   V59A+M284V;     -   A91L+M96I+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S;     -   G108A+M284V;     -   E129V+K177L+R179E;     -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S;     -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+Y276F+L427M;     -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+M284T;     -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+N376*+I377*;     -   E129V+K177L+R179E+K220P+N224L+Q254S;     -   E129V+K177L+R179E+K220P+N224L+Q254S+M284T;     -   E129V+K177L+R179E+S242Q;     -   E129V+K177L+R179E+M284V;     -   E129V+K177L+R179V+K220P+N224L+S242Q+Q254S;     -   K220P+N224L+S242Q+Q254S;     -   K220P+N224L+Q254S;     -   S242Q+M284V;     -   M284V.

In an embodiment the bacterial alpha-amylase variant may be an enzyme having a degree of identity of at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, but less than 100% to any of the sequences shown in SEQ ID NO: 3 in WO 99/19467 or SEQ ID NO: 1 herein.

In an embodiment the bacterial alpha-amylase has a T½ (min) at pH 4.5, 75° C., 0.12 mM CaCl₂) of at least 20, such as at least 25, such as at least 30, such as at least 40, such as at least 50, such as at least 60, such as at least 70, such as at least 80, such as at least 90, such as at least 100, such as at least 110, such as at least 120, such as at least 130, such as at least 140, such as at least 150, such as at least 160, such as at least 170, such as at least 180 such as between 20-300, such as between 50-300, such as between 60-300, such as between 70-300, such as between 80-300, such as between 90-300, such as between 100-300, such as between 120-300, such as between 140-300 such as between 160-300, such as between 180-300.

The bacterial alpha-amylase is typically added in an amount between 0.0005-5 KNU per g DS, preferably between 0.001-1 KNU per g DS, such as around 0.06 KNU per g DS, or 0.0005-5 KNU(S) per g DS, preferably between 0.001-1 KNU(S) per g DS, such as around 0.060 KNU(S) per g DS if it is a Bacillus stearothermophilus alpha-amylase.

Examples of commercial compositions comprising bacterial alpha-amylases include BAN™, TERMAMYL™ SC, LIQUOZYME™ X, LIQUOZYME™ SC, (Novozymes), SPEZYME™ FRED, SPEZYME™ AA, SPEZYME™ DELTA AA, GC358, GC980, and SPEZYME™ RSL (Danisco A/S), and FUELZYME™ from Verenium, USA.

Raw Starch Hydrolyzing Alpha-Amylases

A raw starch hydrolyzing alpha-amylase is present during liquefaction step i) in a process of the invention together with a bacterial alpha-amylase and a carbohydrate-source generating enzyme having a heat stability at 70° C., pH 5.3, of at least 70%, such as at least 75%, preferably at least 80%, preferably at least 85%.

Optionally a protease and/or a pullulanase is present or added during liquefaction as well.

As used herein, a “raw starch hydrolyzing alpha-amylase” refers to an alpha-amylase that can directly degrade raw starch granules below the gelatinization temperature of starch. The gelatinization temperature of starch can range from 51° C. to 78° C. as the gelatinization initiation temperature can vary from about 51° C. to 68° C.

The raw starch hydrolyzing alpha-amylase may be of any origin. In a preferred embodiment the raw starch hydrolyzing alpha-amylase is derived from a fungal organism such as a filamentous fungus.

In an embodiment the raw starch hydrolyzing alpha-amylase is derived from a strain of Aspergillus, such as Aspergillus niger or Aspergillus kawachii.

In a preferred embodiment the fungal acid raw starch hydrolyzing alpha-amylase is a hybrid alpha-amylase.

In an embodiment the raw starch hydrolyzing enzyme is a hybrid enzyme which comprises an amino acid sequence of a catalytic module having alpha-amylase activity and an amino acid sequence of a carbohydrate-binding module, and optionally a linker, wherein the catalytic module is of fungal origin. Specific ensamples of such enzymes are the following enzymes disclosed especially tables 3 and 4 in WO 2005/003311. Raw starch hydrolyzing enzymes include the ones in the following table:

Catalytic Variant module Linker SBD JA001 Aspergillus Aspergillus Aspergillus kawachii AA niger AA kawachii AA (SP288) JA002 SP288 A. kawachii AA Aspergillus niger AMG JA003 SP288 A. kawachii AA Talaromyces emersonii AMG JA004 SP288 A. kawachii AA Athelia rolfsii AMG JA005 SP288 A. kawachii AA Bacillus MA JA007 Aspergillus A. kawachii AA A. kawachii AA kawachii AA JA008 SP288 A. niger AMG A. niger AMG JA009 SP288 A. rolfsii AMG A. niger AMG JA010 SP288 PEPT A. niger AMG JA011 SP288 A. rolfsii AMG A. rolfsii AMG JA012 SP288 A. kawachii AA A. niger AMG + A. rolfsii AMG

In a preferred embodiment the raw starch hydrolyzing enzyme has the Aspergillus niger catalytic domain and an Aspergillus kawachii alpha-amylase (AA) or Athelia rolfsii glucoamylase (AMG) SBD.

In another preferred embodiment the raw starch degrading enzyme is Aspergillus kawachii alpha-amylase.

Specific examples of other contemplated raw starch hydrolyzing hybrid alpha-amylases include those disclosed in WO 2006/069290, especially the Rhizomucor pusillus alpha-amylase with Athelia rolfsii AMG linker and SBD (SEQ ID NO: 101 in U.S. application No. 60/638,614).

In a preferred embodiment the raw starch hydrolyzing alpha-amylase is the hybrid alpha-amylase consisting of Rhizomucor pusillus alpha-amylase with Aspergillus niger glucoamylase linker and SBD disclosed as V039 in Table 5 in WO 2006/069290 (Novozymes A/S).

Also contemplated is the Meripilus giganteus alpha-amylase with Athelia rolfsii glucoamylase linker and SBD (SEQ ID NO: 102 in U.S. application No. 60/638,614).

In another embodiment the raw starch hydrolyzing alpha-amylase is the hybrid alpha-amylase consisting of Meripillus gigantus alpha-amylase disclosed in WO 2006/069290 (Novozymes A/S).

The raw starch hydrolyzing alpha-amylases used in Example 5 herein are variants of the Rhizomucor pusillus alpha-amylase disclosed in U.S. provisional application No. 61/505,192 (which is hereby incorporated by reference).

RSH AA 96 is Rhizomucor pusillus alpha-amylase variant with Aspergillus niger glucoamylase linker and SBD and further the following substitutions: G128D+D143N (using SEQ ID NO: 14 herein for the numbering).

RSH AA 101 is Rhizomucor pusillus alpha-amylase variant with Aspergillus niger glucoamylase linker and SBD and further the following substitutions: G128D+D143N+K192R (using SEQ ID NO: 14 herein for the numbering).

In one embodiment, the raw starch hydrolyzing alpha-amylase is defined as an enzyme that has a raw starch degrading index of at least 0.2, at least 0.3, at least, 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, wherein the raw degrading index is a ratio of activity to degrade raw starch to activity to degrade gelatinized starch (Ra/Ga). Preferably, the raw starch hydrolyzing alpha-amylase is defined as an enzyme that has a raw starch degrading index of higher than 1. The activity on gelatinized starch is measured by measuring the release of glucose produced by the enzyme on a 2% gelatinized (e.g., corn) starch reaction mixture. The activity is measured by the release of reducing sugars produced in 4 mol per hour per mg of pure active enzyme. The same assay can then be used to measure the activity of the enzyme on raw starch, but substituting the 2% gelatinized (e.g., corn) starch by 2% of raw (e.g., corn) starch. In both assays, the temperature is 40° C., the same pH and buffer solution is used and the incubation time is 6 hours, and is further described in the “Materials and Methods” section below.

The raw starch hydrolyzing alpha-amylase for use in the present invention also include alpha-amylases having a high degree of sequence identity to the raw starch hydrolyzing alpha-amylases described herein. In an embodiment, the raw starch hydrolyzing alpha-amylase has sequence identity of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, to the amino acid sequence of a raw starch hydrolyzing alpha-amylases disclosed herein. For example, raw starch hydrolyzing alpha-amylases include raw starch hydrolyzing alpha-amylases having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the hybrid alpha-amylase disclosed as V039 in Table 5 in WO 2006/069290 or SEQ ID NOs: 13 or 14 herein.

In one embodiment, the raw starch hydrolyzing alpha-amylase may be added during liquefaction step i) in an amount of 0.01-1000 micro g Enzyme Protein (EP) per g DS, for example 0.1-500 micro g EP per g DS, such as 1-200 micro g EP per g DS, such as 1-100 micro g EP per g DS.

Carbohydrate-Source Generating Enzymes

According to the invention a carbohydrate-source generating enzyme having a heat stability at 70° C., pH 5.3, of at least 70%, such as at least 75%, preferably at least 80%, preferably at least 85%, preferably a glucoamylase, is present and/or added during liquefaction together with a bacterial alpha-amylase and a raw starch hydrolyzing alpha-amylase. A protease and/or a pullulanase may also be present and/or added during liquefaction step i).

The term “carbohydrate-source generating enzyme” includes any enzymes generating fermentable sugars. A carbohydrate-source generating enzyme is capable of producing a carbohydrate that can be used as an energy-source by the fermenting organism(s) in question, for instance, when used in a process of the invention for producing a fermentation product, such as ethanol. The generated carbohydrates may be converted directly or indirectly to the desired fermentation product, preferably ethanol. According to the invention a mixture of carbohydrate-source generating enzymes may be used. Specific examples include glucoamylase (being glucose generators), beta-amylase and maltogenic amylase (being maltose generators).

In an embodiment the carbohydrate-source generating enzyme, preferably a glucoamylase, has a heat stability at 70° C., pH 5.3, of at least 70%, such as at least 75%, preferably at least 80%, preferably at least 85%.

In an embodiment the carbohydrate-source generating enzyme, preferably a glucoamylase, has a relative activity at pH 4.5 of at least 80%, preferably at least 85%, preferably at least 90% determined as described in Example 4 (pH Optimum).

In an embodiment the carbohydrate-source generating enzyme, preferably a glucoamylase, has a pH stability at pH 4.5 of at least at least 80%, at least 85%, at least 90%, at least 95%, at least 100% determined as described in Example 4 (pH Stability).

In a specific and preferred embodiment the carbohydrate-source generating enzyme is a glucoamylase, preferably of fungal origin, preferably a filamentous fungi, such as from a strain of the genus Penicillium, especially a strain of Penicillium oxalicum disclosed as SEQ ID NO: 2 in PCT/CN10/071753 published as WO 2011/127802 (which is hereby incorporated by reference) and shown in SEQ ID NO: 9 herein or a Penicillium oxalicum variant disclosed in U.S. application No. 61/531,189 or U.S. application No. 61/566,046 or PCT/US12/053779 having a K79V substitution (using SEQ ID NO: 15 herein for numbering). The K79V glucoamylase variant has reduced sensitivity to protease degradation compared to the parent.

In an embodiment the glucoamylase has at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the mature polypeptide shown in SEQ ID NO: 2 in PCT/CN10/071753 published as WO 2011/127802 or SEQ ID NO: 9 herein.

Proteases

According to the invention a protease may be present and/or added during liquefaction in step i) together with a bacterial alpha-amylase, a raw starch hydrolyzing alpha-amylase and a carbohydrate-source generating enzyme, preferably a glucoamylase, having a heat stability at 70° C., pH 5.3, of at least 70%.

The protease may be any protease. In a preferred embodiment the protease is an acid protease of microbial origin, preferably of fungal or bacterial origin. An acid fungal protease is preferred, but also other proteases can be used.

Suitable proteases include microbial proteases, such as fungal and bacterial proteases. Preferred proteases are acidic proteases, i.e., proteases characterized by the ability to hydrolyze proteins under acidic conditions below pH 7.

The acid fungal protease may be derived from Aspergillus, Candida, Coriolus, Endothia, Enthomophtra, Irpex, Mucor, Penicillium, Rhizopus, Sclerotium, and Torulopsis. In particular, the protease may be derived from Aspergillus aculeatus (WO 95/02044), Aspergillus awamori (Hayashida et al., 1977, Agric. Biol. Chem. 42(5), 927-933), Aspergillus niger (see, e.g., Koaze et al., 1964, Agr. Biol. Chem. Japan 28: 216), Aspergillus saitoi (see, e.g., Yoshida, 1954, J. Agr. Chem. Soc. Japan 28: 66), or Aspergillus oryzae, such as the pepA protease; and acidic proteases from Mucor miehei or Mucor pusillus.

The protease may be a neutral or alkaline protease, such as a protease derived from a strain of Bacillus. A particular protease is derived from Bacillus amyloliquefaciens and has the sequence obtainable at Swissprot as Accession No. P06832. The proteases may have at least 90% sequence identity to the amino acid sequence disclosed in the Swissprot Database, Accession No. P06832 such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or particularly at least 99% identity.

The protease may have at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90% sequence identity to the amino acid sequence disclosed as SEQ ID NO: 1 in WO 2003/048353 or SEQ ID NO: 3 herein, such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or particularly at least 99% identity.

The protease may be a papain-like protease selected from the group consisting of proteases within EC 3.4.22.* (cysteine protease), such as EC 3.4.22.2 (papain), EC 3.4.22.6 (chymopapain), EC 3.4.22.7 (asclepain), EC 3.4.22.14 (actinidain), EC 3.4.22.15 (cathepsin L), EC 3.4.22.25 (glycyl endopeptidase) and EC 3.4.22.30 (caricain).

In an embodiment, the protease is a protease preparation derived from a strain of Aspergillus, such as Aspergillus oryzae. In another embodiment the protease is derived from a strain of Rhizomucor, preferably Rhizomucor miehei. In another embodiment the protease is a protease preparation, preferably a mixture of a proteolytic preparation derived from a strain of Aspergillus, such as Aspergillus oryzae, and a protease derived from a strain of Rhizomucor, preferably Rhizomucor miehei.

Aspartic acid proteases are described in, for example, Handbook of Proteolytic Enzymes, Edited by A. J. Barrett, N. D. Rawlings and J. F. Woessner, Academic Press, San Diego, 1998, Chapter 270. Examples of aspartic acid proteases include, e.g., those disclosed in Berka et al., 1990, Gene 96: 313; Berka et al., 1993, Gene 125: 195-198; and Gomi et al., 1993, Biosci. Biotech. Biochem. 57: 1095-1100, which are hereby incorporated by reference.

The protease also may be a metallo protease, which is defined as a protease selected from the group consisting of:

-   (a) proteases belonging to EC 3.4.24 (metalloendopeptidases);     preferably EC 3.4.24.39 (acid metallo proteinases); -   (b) metallo proteases belonging to the M group of the above     Handbook; -   (c) metallo proteases not yet assigned to clans (designation: Clan     MX), or belonging to either one of clans MA, MB, MC, MD, ME, MF, MG,     MH (as defined at pp. 989-991 of the above Handbook); -   (d) other families of metallo proteases (as defined at pp. 1448-1452     of the above Handbook); -   (e) metallo proteases with a HEXXH motif; -   (f) metallo proteases with an HEFTH motif; -   (g) metallo proteases belonging to either one of families M3, M26,     M27, M32, M34, M35, M36, M41, M43, or M47 (as defined at pp.     1448-1452 of the above Handbook); -   (h) metallo proteases belonging to the M28E family; and -   (i) metallo proteases belonging to family M35 (as defined at pp.     1492-1495 of the above Handbook).

In other particular embodiments, metallo proteases are hydrolases in which the nucleophilic attack on a peptide bond is mediated by a water molecule, which is activated by a divalent metal cation. Examples of divalent cations are zinc, cobalt or manganese. The metal ion may be held in place by amino acid ligands. The number of ligands may be five, four, three, two, one or zero. In a particular embodiment the number is two or three, preferably three.

There are no limitations on the origin of the metallo protease used in a process of the invention. In an embodiment the metallo protease is classified as EC 3.4.24, preferably EC 3.4.24.39. In one embodiment, the metallo protease is an acid-stable metallo protease, e.g., a fungal acid-stable metallo protease, such as a metallo protease derived from a strain of the genus Thermoascus, preferably a strain of Thermoascus aurantiacus, especially Thermoascus aurantiacus CGMCC No. 0670 (classified as EC 3.4.24.39). In another embodiment, the metallo protease is derived from a strain of the genus Aspergillus, preferably a strain of Aspergillus oryzae.

In one embodiment the metallo protease has a degree of sequence identity to amino acids −178 to 177, −159 to 177, or preferably amino acids 1 to 177 (the mature polypeptide) of SEQ ID NO: 1 of WO 2010/008841 or SEQ ID NO: 3 herein (a Thermoascus aurantiacus metallo protease) of at least 80%, at least 82%, at least 85%, at least 90%, at least 95%, or at least 97% such as at least 98%, such as at least 99%; and which have metallo protease activity. In particular embodiments, the metallo protease consists of an amino acid sequence with a degree of identity to SEQ ID NO: 1 as mentioned above or shown as SEQ ID NO: 3 herein.

The Thermoascus aurantiacus metallo protease is a preferred example of a metallo protease suitable for use in a process of the invention. Another metallo protease is derived from Aspergillus oryzae and comprises the sequence of SEQ ID NO: 11 disclosed in WO 2003/048353, or amino acids −23-353; −23-374; −23-397; 1-353; 1-374; 1-397; 177-353; 177-374; or 177-397 thereof, and SEQ ID NO: 10 disclosed in WO 2003/048353.

Another metallo protease suitable for use in a process of the invention is the Aspergillus oryzae metallo protease comprising SEQ ID NO: 5 of WO 2010/008841, or a metallo protease is an isolated polypeptide which has a degree of identity to SEQ ID NO: 5 of at least about 80%, at least 82%, at least 85%, at least 90%, at least 95%, or at least 97%; and which have metalloprotease activity. In particular embodiments, the metallo protease consists of the amino acid sequence of SEQ ID NO: 5 of WO 2010/008841 (hereby incorporated by reference).

In a particular embodiment, a metallo protease has an amino acid sequence that differs by forty, thirty-five, thirty, twenty-five, twenty, or by fifteen amino acids from amino acids −178 to 177, −159 to 177, or +1 to 177 of the amino acid sequences of the Thermoascus aurantiacus or Aspergillus oryzae metallo protease.

In another embodiment, a metallo protease has an amino acid sequence that differs by ten, or by nine, or by eight, or by seven, or by six, or by five amino acids from amino acids −178 to 177, −159 to 177, or +1 to 177 of the amino acid sequences of these metallo proteases, e.g., by four, by three, by two, or by one amino acid.

In particular embodiments, the metallo protease a) comprises or b) consists of

-   i) the amino acid sequence of amino acids −178 to 177, −159 to 177,     or +1 to 177 of SEQ ID NO:1 of WO 2010/008841; -   ii) the amino acid sequence of amino acids −23-353, −23-374,     −23-397, 1-353, 1-374, 1-397, 177-353, 177-374, or 177-397 of SEQ ID     NO: 3 of WO 2010/008841; -   iii) the amino acid sequence of SEQ ID NO: 5 of WO 2010/008841; or     allelic variants, or fragments, of the sequences of i), ii),     and iii) that have protease activity.

A fragment of amino acids −178 to 177, −159 to 177, or +1 to 177 of SEQ ID NO: 1 of WO 2010/008841 or of amino acids −23-353, −23-374, −23-397, 1-353, 1-374, 1-397, 177-353, 177-374, or 177-397 of SEQ ID NO: 3 of WO 2010/008841; is a polypeptide having one or more amino acids deleted from the amino and/or carboxyl terminus of these amino acid sequences. In one embodiment a fragment contains at least 75 amino acid residues, or at least 100 amino acid residues, or at least 125 amino acid residues, or at least 150 amino acid residues, or at least 160 amino acid residues, or at least 165 amino acid residues, or at least 170 amino acid residues, or at least 175 amino acid residues.

In another embodiment, the metallo protease is combined with another protease, such as a fungal protease, preferably an acid fungal protease.

Commercially available products include ALCALASE®, ESPERASE™ FLAVOURZYME™, NEUTRASE®, RENNILASE®, NOVOZYM™ FM 2.0L, and iZyme BA (available from Novozymes A/S, Denmark) and GC106™ and SPEZYME™ FAN from Genencor International, Inc., USA.

The protease may be present in an amount of 0.0001-1 mg enzyme protein per g DS, preferably 0.001 to 0.1 mg enzyme protein per g DS. Alternatively, the protease may be present in an amount of 0.0001 to 1 LAPU/g DS, preferably 0.001 to 0.1 LAPU/g DS and/or 0.0001 to 1 mAU-RH/g DS, preferably 0.001 to 0.1 mAU-RH/g DS.

In an embodiment the protease used in a process of the invention is a thermostable protease. Preferably one disclosed in WO 2011/072191 (hereby incorporated by reference) which may have either

-   i) a thermostability value of more than 20% determined as Relative     Activity at 80° C./70° C.; and/or -   ii) a thermostability value of more than 10% determined as Relative     Activity at 85° C./70° C.

In an embodiment the protease has a thermostability value:

-   -   of more than 30%, more than 40%, more than 50%, more than 60%,         more than 70%, more than 80%, more than 90% determined as         Relative Activity at 80° C./70° C., and/or     -   of more than 12%, more than 14%, more than 16%, more than 18%,         more than 20%, determined as Relative Activity at 85° C./70° C.;         and/or     -   of more than 20%, more than 30%, more than 40%, more than 50%,         more than 60%, more than 70%, more than 80%, more than 90%         determined as Remaining Activity at 80° C.; and/or of more that         20%, more than 30%, more than 40%, more than 50%, more than 60%,         more than 70%, more than 80%, more than 90% determined as         Remaining Activity at 84° C. and/or.

Purified variants may have a themostability for above 90, above 100 at 85° C. as determined using the Zein-BCA assay as disclosed in Example 3.

Determination of “Relative Activity” and “Remaining Activity” is determined as described in Example 2.

In a preferred embodiment the thermostable protease used in a process of the invention is a “metallo protease” defined as a protease belonging to EC 3.4.24 (metalloendopeptidases); preferably EC 3.4.24.39 (acid metallo proteinases).

To determine whether a given protease is a metallo protease or not, reference is made to the above “Handbook of Proteolytic Enzymes” and the principles indicated therein. Such determination can be carried out for all types of proteases, be it naturally occurring or wild-type proteases; or genetically engineered or synthetic proteases.

Protease activity can be measured using any suitable assay, in which a substrate is employed, that includes peptide bonds relevant for the specificity of the protease in question. Assay-pH and assay-temperature are likewise to be adapted to the protease in question. Examples of assay-pH-values are pH 6, 7, 8, 9, 10, or 11. Examples of assay-temperatures are 30, 35, 37, 40, 45, 50, 55, 60, 65, 70 or 80° C.

Examples of protease substrates are casein, such as Azurine-Crosslinked Casein (AZCL-casein). Two protease assays are described below in the “Materials & Methods”-section, of which the so-called “AZCL-Casein Assay” is the preferred assay.

In an embodiment the protease has at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 100% of the activity of the JTP196 protease variant or Protease Pfu determined by the AZCL-casein assay.

There are no limitations on the origin of the thermostable protease used in a process of the invention as long as it fulfills the thermostability properties defined above. The protease may be a variant of, e.g., a wild-type protease as long as the protease has the thermostability properties defined above. In a preferred embodiment the protease is a variant of a metallo protease as defined above. In an embodiment the protease used in a process of the invention is of fungal origin, such as a fungal metallo protease, such as a fungal metallo protease derived from a strain of the genus Thermoascus, preferably a strain of Thermoascus aurantiacus, especially Thermoascus aurantiacus CGMCC No. 0670 (classified as EC 3.4.24.39).

In an embodiment the protease is a variant of the mature part of the metallo protease shown in SEQ ID NO: 2 disclosed in WO 2003/048353 or the mature part of SEQ ID NO: 1 in WO 2010/008841 and shown as SEQ ID NO: 3 herein with the following mutations:

-   -   S5*+N26R+D79L+S87P+A112P+D142L;     -   S5*+D79L+S87P+A112P+D142L;     -   N26R+T46R+D79L+S87P+A112P+D142L;     -   A27K+D79L+Y82F+S87G+D104P+A112P+A126V+D142L;     -   A27K+D79L+Y82F+D104P+A112P+A126V+D142L;     -   A27K+Y82F+S87G+D104P+A112P+A126V+D142L;     -   A27K+D79L+S87P+A112P+T124V+D142L;     -   A27K+D79L+S87P+A112P+A126V+D142L;     -   A27K+D79L+S87P+A112P+D142L.     -   A27K+Y82F+D104P+A112P+A126V+D142L;     -   S36P+D79L+S87P+A112P+D142L;     -   A37P+D79L+S87P+A112P+D142L;     -   S38T+D79L+S87P+A112P+A126V+D142L;     -   T46R+D79L+S87P+T116V+D142L;     -   S49P+D79L+S87P+A112P+D142L;     -   S50P+D79L+S87P+A112P+D142L;     -   S70V+D79L+Y82F+S87G+Y97W+A112P+D142L;     -   S70V+D79L+Y82F+S87G+A112P+D142L;     -   D79L+P81R+S87P+A112P+D142L;     -   D79L+Y82F+S87G+Y97W+D104P+A112P+D142L;     -   D79L+Y82F+S87G+D104P+A112P+D142L;     -   D79L+Y82F+S87G+A112P+A126V+D142L;     -   D79L+Y82F+S87G+A112P+D142L;     -   D79L+Y82F+S87P+A112P+T124V+D142L;     -   D79L+Y82F+S87P+A112P+A126V+D142L;     -   D79L+Y82F+S87P+A112P+D142L;     -   D79L+S87P+N98C+A112P+G135C+D142L;     -   D79L+S87P+D104P+A112P+D142L;     -   D79L+S87P+A112P+T124V+A126V+D142L;     -   D79L+S87P+A112P+T124V+D142L;     -   D79L+S87P+A112P+D142L;     -   D79L+S87P+A112P+D142L+T141C+M161C;     -   Y82F+S87G+S70V+D79L+D104P+A112P+D142L;     -   Y82F+S87G+D79L+D104P+A112P+A126V+D142L.

In an embodiment the thermostable protease variant has at least 75% identity preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% identity to the mature part of the polypeptide of SEQ ID NO: 2 disclosed in WO 2003/048353 or the mature part of SEQ ID NO: 1 in WO 2010/008841 or SEQ ID NO: 3 herein.

In an embodiment the protease is derived from a strain of Pyrococcus, such as a strain of Pyrococcus furiosus. In an embodiment the protease is the one shown as SEQ ID NO: 1 in U.S. Pat. No. 6,358,726-B1 (Takara Shuzo Company). In another embodiment the protease is one disclosed in SEQ ID NO: 16 herein or a protease having at least 80% identity, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to SEQ ID NO: 1 in U.S. Pat. No. 6,358,726-B1 or SEQ ID NO: 16 herein. The Pyroccus furiosus protease can be purchased from Takara Bio, Japan.

The Pyrococcus furiosus protease is a thermostable protease. The commercial Pyrococcus furiosus protease product (Pfu S) from Takara Bio, Japan was found to have a thermostability of 110% (80° C./70° C.) and 103% (90° C./70° C.) at pH 4.5 determined as described in Example 2 herein.

Pullulanase

According to the invention a pullulanase may further be present during liquefaction, saccharification and/or fermentation.

In an embodiment a pullulanase is present and/or added during liquefaction step i).

In another embodiment a pullulanase is present and/or added during saccharification or simultaneous saccharification and fermentation (SSF).

Pullulanases (E.C. 3.2.1.41, pullulan 6-glucano-hydrolase), are debranching enzymes characterized by their ability to hydrolyze the alpha-1,6-glycosidic bonds in, for example, amylopectin and pullulan.

The pullulanase may be any pullulanase. In an embodiment the pullulanase is a bacterial pullulanase, especially a pullulanase derived from a strain on the genus Bacillus, especially derived from a strain of Bacillus deramificans. EP 605,040 discloses such pullulanase derived from Bacillus deramificans.

In an embodiment the pullulanase is a variant disclosed in WO 00/01796. Contemplated pullulanases include the pullulanases from Bacillus amyloderamificans disclosed in U.S. Pat. No. 4,560,651 (hereby incorporated by reference), the pullulanase disclosed as SEQ ID NO: 2 in WO 01/151620 (hereby incorporated by reference), the Bacillus deramificans disclosed as SEQ ID NO: 4 in WO 01/151620 (hereby incorporated by reference), and the pullulanase from Bacillus acidopullulyticus disclosed as SEQ ID NO: 6 in WO 01/151620 (hereby incorporated by reference) and also described in FEMS Mic. Let. 115: 97-106 (1994).

Additional pullulanases contemplated according to the present invention included the pullulanases from Pyrococcus woesei, specifically from Pyrococcus woesei DSM No. 3773 disclosed in WO 92/02614.

In an embodiment the pullulanase is a family GH57 pullulanase. In an embodiment the pullulanase includes an X47 domain as disclosed in WO 2011/087836 (which are hereby incorporated by reference). More specifically the pullulanase may be derived from a strain of the genus Thermococcus, including Thermococcus litoralis and Thermococcus hydrothermalis, such as the Thermococcus hydrothermalis pullulanase shown in SEQ ID NO: 11 truncated at site X4 right after the X47 domain (i.e., amino acids 1-782 in SEQ ID NOS: 11 and 12). The pullulanase may also be a hybrid of the Thermococcus litoralis and Thermococcus hydrothermalis pullulanases or a T. hydrothermalis/T. litoralis hybrid enzyme with truncation site X4 disclosed in WO 2011/087836 (which is hereby incorporated by reference) or disclosed in SEQ ID NO: 12 herein.

In another embodiment the pullulanase is derived from a strain of Thermococcus, such as especially Thermococcus hydrothermalis. In an embodiment the pullulanase is a variant of Thermococcus hydrothermalis. In an embodiment the pullulanase comprises X47 domain. In an embodiment the pullulanase is truncated, such as one disclosed in WO 2011/087836. In an embodiment the pullulanase comprises an X46 domain such as one disclosed in WO 2011/076123.

The pullulanases added during liquefaction and saccharification and/or fermentation may not be the same. For instance, in an embodiment the pullulanase present and/or added during liquefaction step i) is derived from Thermococcus hydrothermalis, while the pullulanase optionally added during saccharification and/or fermentation is derived from Bacillus deramificans. Pullulanase activity may be determined as NPUN. An Assay for determination of NPUN is described in the “Materials & Methods”-section below.

The pullulanase may according to the invention be added in an effective amount which include the preferred amount of about 0.0001-10 mg enzyme protein per gram DS, preferably 0.0001-0.10 mg enzyme protein per gram DS, more preferably 0.0001-0.010 mg enzyme protein per gram DS. Pullulanase activity may be determined as NPUN. An Assay for determination of NPUN is described in the “Materials & Methods”-section below.

Commercially available pullulanase products include PROMOZYME D, PROMOZYME™ D2 (Novozymes A/S, Denmark), OPTIMAX L-300 (Danisco, USA), and AMANO 8 (Amano, Japan).

Carbohydrate-Source Generating Enzyme Present and/or Added During Saccharification and/or Fermentation

According to the invention a carbohydrate-source generating enzyme, preferably a glucoamylase, may be present and/or added during saccharification and/or fermentation. The carbohydrate-source generating enzyme may be difference from the carbohydrate-source generating enzyme, preferably glucoamylase, present and/or added during liquefaction step i).

Glucoamylase

According to the invention the glucoamylase present and/or added during saccharification and/or fermentation may be derived from any suitable source, e.g., derived from a microorganism or a plant. Preferred glucoamylases are of fungal or bacterial origin, selected from the group consisting of Aspergillus glucoamylases, in particular Aspergillus niger G1 or G2 glucoamylase (Boel et al., 1984, EMBO J. 3(5): 1097-1102), or variants thereof, such as those disclosed in WO 92/00381, WO 00/04136 and WO 01/04273 (from Novozymes, Denmark); the A. awamori glucoamylase disclosed in WO 84/02921, Aspergillus oryzae glucoamylase (Agric. Biol. Chem. 55(4): 941-949 (1991)), or variants or fragments thereof. Other Aspergillus glucoamylase variants include variants with enhanced thermal stability: G137A and G139A (Chen et al., 1996, Prot. Eng. 9: 499-505); D257E and D293E/Q (Chen et al., 1995, Prot. Eng. 8: 575-582); N182 (Chen et al., 1994, Biochem. J. 301: 275-281); disulphide bonds, A246C (Fierobe et al., 1996, Biochemistry 35: 8698-8704; and introduction of Pro residues in position A435 and S436 (Li et al., 1997, Protein Eng. 10: 1199-1204.

Other glucoamylases include Athelia rolfsii (previously denoted Corticium rolfsii) glucoamylase (see U.S. Pat. No. 4,727,026 and (Nagasaka et al., 1998, “Purification and properties of the raw-starch-degrading glucoamylases from Corticium rolfsii, Appl. Microbiol. Biotechnol. 50:323-330), Talaromyces glucoamylases, in particular derived from Talaromyces emersonii (WO 99/28448), Talaromyces leycettanus (U.S. Pat. No. RE32,153), Talaromyces duponti, Talaromyces thermophilus (U.S. Pat. No. 4,587,215). In a preferred embodiment the glucoamylase used during saccharification and/or fermentation is the Talaromyces emersonii glucoamylase disclosed in WO 99/28448.

In an embodiment the glucoamylase is a glucoamylase which exhibits at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% identity to the mature enzyme sequence shown as SEQ ID NO: 7 in WO 99/28448.

Bacterial glucoamylases contemplated include glucoamylases from the genus Clostridium, in particular C. thermoamylolyticum (EP 135,138), and C. thermohydrosulfuricum (WO 86/01831) and Trametes cingulata, Pachykytospora papyracea; and Leucopaxillus giganteus all disclosed in WO 2006/069289; or Peniophora rufomarginata disclosed in WO 2007/124285; or a mixture thereof. Also hybrid glucoamylase are contemplated according to the invention. Examples the hybrid glucoamylases disclosed in WO 2005/045018. Specific examples include the hybrid glucoamylase disclosed in Table 1 and 4 of Example 1 (which hybrids are hereby incorporated by reference).

In an embodiment the glucoamylase is derived from a strain of the genus Pycnoporus, in particular a strain of Pycnoporus as described in WO 2011/066576 (Novozymes), or from a strain of the genus Gloephyllum, in particular a strain of Gloephyllum as described in WO 2011/068803 (Novozymes) or a strain of the genus Nigrofomes, in particular a strain of Nigrofomes sp. disclosed in PCT/US10/058375 published as WO 2012/064351 (Novozymes).

Contemplated are also glucoamylases which exhibit a high identity to any of above mention glucoamylases, i.e., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% identity to the mature enzymes sequences mentioned above.

In an embodiment the glucoamylase is a glucoamylase which exhibits at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% identity to the mature enzyme sequences shown in any one of SEQ ID NO: 2, 4 or 6 in WO 2011/066576.

In an embodiment the glucoamylase is a glucoamylase which exhibits at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% identity to the mature enzyme sequences shown in any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 or 18 in WO 2011/068803.

Carbohydrase-source generating enzymes, such as preferably glucoamylases, may in an embodiment be added in an amount of 0.0001-20 AGU/g DS, preferably 0.001-10 AGU/g DS, especially between 0.01-5 AGU/g DS, such as 0.1-2 AGU/g DS.

Commercially available compositions comprising glucoamylase include AMG 200L; AMG 300 L; SANT™ SUPER, SAN™ EXTRA L, SPIRIZYME™ PLUS, SPIRIZYME™ FUEL, SPIRIZYME™ B4U, SPIRIZYME™ ULTRA and AMG™ E (from Novozymes A/S); OPTIDEX™ 300, GC480, GC417 (from Genencor Int.); AMIGASE™ and AMIGASE™ PLUS (from DSM); G-ZYME™ G900, G-ZYME™ and G990 ZR (from Genencor Int.).

Maltoqenic Amylase

The carbohydrate-source generating enzyme present and/or added during saccharification and/or fermentation may also be a maltogenic alpha-amylase. A “maltogenic alpha-amylase” (glucan 1,4-alpha-maltohydrolase, E.C. 3.2.1.133) is able to hydrolyze amylose and amylopectin to maltose in the alpha-configuration. A maltogenic amylase from Bacillus stearothermophilus strain NCIB 11837 is commercially available from Novozymes A/S. Maltogenic alpha-amylases are described in U.S. Pat. Nos. 4,598,048, 4,604,355 and 6,162,628, which are hereby incorporated by reference.

The maltogenic amylase may in a preferred embodiment be added in an amount of 0.05-5 mg total protein/gram DS or 0.05-5 MANU/g DS.

Composition Comprising Bacterial Alpha-Amylase, Raw Starch Hydrolyzing Alpha-amylase and Carbohydrate-Source Generating Enzyme.

Compositions of the invention may be added during liquefaction step i) in a process of the invention. Compositions of the invention comprise a bacterial alpha-amylase, a raw starch hydrolyzing alpha-amylase and carbohydrate-source generating enzyme. The composition may further comprise a protease and/or a pullulanase and other enzymes.

Thus, in this aspect the invention relates to composition comprising

-   -   a bacterial alpha-amylase;     -   a raw starch hydrolyzing alpha-amylase;     -   carbohydrate-source generating enzyme having a heat stability at         70° C., pH 5.3, of at least 70%.

In an embodiment the bacterial alpha-amylase is derived from a starch of Bacillus. Suitable bacterial alpha-amylases are described in the “Bacterial Alpha-Amylase” section above.

In an embodiment the bacterial alpha-amylase has at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity to the mature part of the polypeptide of SEQ ID NO: 3 disclosed in WO 99/019467 or SEQ ID NO: 1 herein.

In a preferred embodiment the bacterial alpha-amylase is derived from Bacillus stearothermophilus alpha-amylases, especially a variant which comprising have a double deletion corresponding to a deletion of positions 181 and 182 and further comprise a N193F substitution (also denoted I181*+G182*+N193F). The Bacillus stearothermophilus alpha-amylases may be a variants disclosed in disclosed in WO 2011/082425 or below specifically disclosed.

Preferred bacterial alpha-amylases are derived from the Bacillus stearothermophilus alpha-amylase shown in SEQ ID NO: 1 herein truncated to have about 491 amino acids with the mutations selected from the group consisting of:

-   -   V59A+Q89R+E129V+K177L+R179E+I181*+G182*+N193F+H208Y+K220P+N224L+Q254S;     -   E129V+K177L+R179E+I181*+G182*+N193F; and     -   E129V+K177L+R179E+I181*+G182*+N193F+K220P+N224L+S242Q+Q254S.

In an embodiment the bacterial alpha-amylase variant has at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% identity to the mature part of the polypeptide of SEQ ID NO: 3 disclosed in WO 99/019467 or SEQ ID NO: 1 herein.

Suitable raw starch hydrolyzing enzymes are described in the “Raw Starch Hydrolyzing Alpha-Amylase” section above.

In an embodiment the raw starch hydrolyzing alpha-amylase is of fungal origin. In a preferred embodiment the raw starch hydrolyzing alpha-amylase is a variant of Rhizomucor pusillus alpha-amylase with Aspergillus niger glucoamylase linker and SBD and further one or more of the following substitutions: G128D, D143N, K192R, such as especially G128D+D143N or G128D+D143N+K192R (using SEQ ID NO: 14 herein for the numbering).

Suitable carbohydrate-source generating enzymes, preferably glucoamylases, are described in the “Carbohydrate-Source Generating Enzymes” section above.

In an embodiment the carbohydrate-source generating enzyme is a glucoamylase having a heat stability at 70° C., pH 5.3, of at least 70% such as at least 75%, preferably at least 80%, preferably at least 85, is a glucoamylase. In a preferred embodiment the glucoamylase is from the genus Penicillium, especially a strain of Penicillium oxalicum disclosed as SEQ ID NO: 2 in PCT/CN10/071753 published as WO 2011/127802 (which is hereby incorporated by reference) and shown in SEQ ID NO: 9 herein or a protease stable protein engineered variant(s) of the Penicillium oxalicum glucoamylase disclosed in U.S. application No. 61/531,189 or U.S. application No. 61/566,046 or PCT/US12/053779 (Novozymes) having a K79V substitution (using SEQ ID NO: 15 herein for numbering).

In another embodiment the carbohydrate-source generating enzyme is the glucoamylase shown in SEQ ID NO: 9 or a glucoamylase having at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity SEQ ID NO: 9 herein.

In an embodiment the composition further comprises a protease. The protease may be of fungal or bacterial origin. Suitable proteases are described in the “Protease” section above.

In an embodiment the protease is a metallo protease. In an embodiment the protease is derived from the genus Thermoascus, preferably a strain of Thermoascus aurantiacus, especially Thermoascus aurantiacus CGMCC No. 0670 (classified as EC 3.4.24.39) disclosed in SEQ ID NO: 3 herein or amino acids 1 to 177 (the mature polypeptide) of SEQ ID NO: 1 of WO 2010/008841.

In an embodiment protease is derived from a strain of Pyrococcus, preferably a strain of Pyrococcus furiosus, such as the one shown in SEQ ID NO: 1 in U.S. Pat. No. 6,258,726 or SEQ ID NO: 16 herein.

In an embodiment the protease is one having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to in SEQ ID NO: 1 in U.S. Pat. No. 6,258,726 or SEQ ID NO: 16 herein.

In an embodiment the composition of the invention comprises

-   -   a bacterial alpha-amylase derived from Bacillus         stearothermophilus;     -   a raw starch hydrolyzing alpha-amylase derived from Rhizomucor         pusillus;     -   a carbohydrate-source generating enzyme having a heat stability         at 70° C., pH 5.3, of at least 70% derived from Penicillium         oxalicum.

In an embodiment the composition further comprises a protease derived from Thermoascus aurantiacus or Pyrococcus furiosus.

In an embodiment the composition further comprises a pullulanase. Suitable pullulanases are described in the “Pullulanase”-section above.

In an embodiment the pullulanase is derived from the genus Thermococcus, such as a strain of Thermococcus hydrothermalis pullulanase shown in SEQ ID NO: 11 truncated right after the X47 domain (i.e., amino acids 1-782 in SEQ ID NO: 11).

Materials & Methods

Materials:

Reference Alpha-Amylase A:

Bacillus stearothermophilus alpha-amylase with the mutations I181*+G182*+N193F truncated to 491 amino acids (using SEQ ID NO: 1 herein for numbering)

Alpha-Amylase (BAA 1407):

Bacillus stearothermophilus alpha-amylase with the mutations: V59A+Q89R+E129V+K177L+R179E+I181*+G182*+N193F+H208Y+K220P+N224L+Q254S truncated to 491 amino acids (using SEQ ID NO: 1 herein for numbering).

Alpha-Amylase (RSH AA 96):

Hybrid alpha-amylase consisting of Rhizomucor pusillus alpha-amylase with Aspergillus niger glucoamylase linker and SBD disclosed as V039 in Table 5 in WO 2006/069290 (Novozymes A/S) or SEQ ID NO: 14 herein with the following substitutions: G128D+D143N.

Alpha-Amylase (RSH AA 101):

Hybrid alpha-amylase consisting of Rhizomucor pusillus alpha-amylase with Aspergillus niger glucoamylase linker and SBD disclosed as V039 in Table 5 in WO 2006/069290 (Novozymes A/S) or SEQ ID NO: 14 herein with the following substitutions: G128D+D143N+K192R.

Glucoamylase (AMG 001):

Mature part of the Penicillium oxalicum glucoamylase disclosed as SEQ ID NO: 2 in PCT/CN10/071753 published as WO 2011/127802 and shown in SEQ ID NO: 9 and 15 herein with a K79V substitution (using SEQ ID NO: 15 herein for numbering) as disclosed in U.S. application No. 61/531,189.

Glucoamylase (AMG SPU):

Tamaromyces emersonii glucoamylase disclosed in WO 99/28448 with about 20% glucoamylase activity from Trametes cingulata glucoamylase disclosed in WO 06/069289 and side activity from hybrid alpha-amylase consisting of Rhizomucor pusillus alpha-amylase with Aspergillus niger glucoamylase linker and SBD disclosed as V039 in Table 5 in WO 2006/069290 (Novozymes A/S) or SEQ ID NO: 14 herein.

Corn:

Ground corn and backset used in Example 5 were obtained from Corn LP in November of 2010 The dry solids (% DS) contents of the ground corn and backset were measured to be 86.78 and 7.93%, respectively, by oven drying at 105° C. for 3 hours.

Yeast:

RED STAR ETHANOL RED™ available from Red Star/Lesaffre, USA.

Methods

Identity:

The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “identity”.

For purposes of the present invention the degree of identity between two amino acid sequences, as well as the degree of identity between two nucleotide sequences, may be determined by the program “align” which is a Needleman-Wunsch alignment (i.e. a global alignment). The program is used for alignment of polypeptide, as well as nucleotide sequences. The default scoring matrix BLOSUM50 is used for polypeptide alignments, and the default identity matrix is used for nucleotide alignments. The penalty for the first residue of a gap is −12 for polypeptides and −16 for nucleotides. The penalties for further residues of a gap are −2 for polypeptides, and −4 for nucleotides.

“Align” is part of the FASTA package version v20u6 (see Pearson and Lipman, 1988, “Improved Tools for Biological Sequence Analysis”, PNAS 85:2444-2448, and Pearson, 1990, “Rapid and Sensitive Sequence Comparison with FASTP and FASTA,” Methods in Enzymology 183:63-98). FASTA protein alignments use the Smith-Waterman algorithm with no limitation on gap size (see “Smith-Waterman algorithm”, Smith and Waterman, 1981, J. Mol. Biol. 147:195-197).

Protease Assays

AZCL-Casein Assay

A solution of 0.2% of the blue substrate AZCL-casein is suspended in Borax/NaH₂PO₄ buffer pH9 while stirring. The solution is distributed while stirring to microtiter plate (100 microL to each well), 30 microL enzyme sample is added and the plates are incubated in an Eppendorf Thermomixer for 30 minutes at 45° C. and 600 rpm. Denatured enzyme sample (100° C. boiling for 20 min) is used as a blank. After incubation the reaction is stopped by transferring the microtiter plate onto ice and the coloured solution is separated from the solid by centrifugation at 3000 rpm for 5 minutes at 4° C. 60 microL of supernatant is transferred to a microtiter plate and the absorbance at 595 nm is measured using a BioRad Microplate Reader.

pNA-Assay

50 microL protease-containing sample is added to a microtiter plate and the assay is started by adding 100 microL 1 mM pNA substrate (5 mg dissolved in 100 microL DMSO and further diluted to 10 mL with Borax/NaH₂PO₄ buffer pH 9.0). The increase in OD₄₀₅ at room temperature is monitored as a measure of the protease activity.

Glucoamylase Activity (AGU)

Glucoamylase activity may be measured in Glucoamylase Units (AGU).

The Novo Glucoamylase Unit (AGU) is defined as the amount of enzyme, which hydrolyzes 1 micromole maltose per minute under the standard conditions 37° C., pH 4.3, substrate: maltose 23.2 mM, buffer: acetate 0.1 M, reaction time 5 minutes.

An autoanalyzer system may be used. Mutarotase is added to the glucose dehydrogenase reagent so that any alpha-D-glucose present is turned into beta-D-glucose. Glucose dehydrogenase reacts specifically with beta-D-glucose in the reaction mentioned above, forming NADH which is determined using a photometer at 340 nm as a measure of the original glucose concentration.

AMG incubation: Substrate: maltose 23.2 mM Buffer: acetate 0.1M pH: 4.30 ± 0.05 Incubation temperature: 37° C. ± 1 Reaction time: 5 minutes Enzyme working range: 0.5-4.0 AGU/mL

Color reaction: GlucDH: 430 U/L Mutarotase: 9 U/L NAD: 0.21 mM Buffer: phosphate 0.12M; 0.15M NaCl pH: 7.60 ± 0.05 Incubation temperature: 37° C. ± 1 Reaction time: 5 minutes Wavelength: 340 nm

A folder (EB-SM-0131.02/01) describing this analytical method in more detail is available on request from Novozymes A/S, Denmark, which folder is hereby included by reference.

Alpha-Amylase Activity (KNU)

The alpha-amylase activity may be determined using potato starch as substrate. This method is based on the break-down of modified potato starch by the enzyme, and the reaction is followed by mixing samples of the starch/enzyme solution with an iodine solution. Initially, a blackish-blue color is formed, but during the break-down of the starch the blue color gets weaker and gradually turns into a reddish-brown, which is compared to a colored glass standard.

One Kilo Novo alpha amylase Unit (KNU) is defined as the amount of enzyme which, under standard conditions (i.e., at 37° C. +/−0.05; 0.0003 M Ca²⁺; and pH 5.6) dextrinizes 5260 mg starch dry substance Merck Amylum solubile.

A folder EB-SM-0009.02/01 describing this analytical method in more detail is available upon request to Novozymes A/S, Denmark, which folder is hereby included by reference.

KNU(S) Alpha-amylase Activity

KNU(S) is used to determine the activity of Bacillus stearothermophilus alpha-amylase and is described on page 35-41 in WO 99/19467 (hereby incorporated by reference).

Determination of Pullulanase Activity (NPUN)

Endo-pullulanase activity in NPUN is measured relative to a Novozymes pullulanase standard. One pullulanase unit (NPUN) is defined as the amount of enzyme that releases 1 micro mol glucose per minute under the standard conditions (0.7% red pullulan (Megazyme), pH 5, 40° C., 20 minutes). The activity is measured in NPUN/ml using red pullulan.

1 mL diluted sample or standard is incubated at 40° C. for 2 minutes. 0.5 mL 2% red pullulan, 0.5 M KCl, 50 mM citric acid, pH 5 are added and mixed. The tubes are incubated at 40° C. for 20 minutes and stopped by adding 2.5 ml 80% ethanol. The tubes are left standing at room temperature for 10-60 minutes followed by centrifugation 10 minutes at 4000 rpm. OD of the supernatants is then measured at 510 nm and the activity calculated using a standard curve.

The present invention is described in further detail in the following examples which are offered to illustrate the present invention, but not in any way intended to limit the scope of the invention as claimed. All references cited herein are specifically incorporated by reference for that which is described therein.

Raw Starch Degrading Enzyme (Ra/Ga) Assay

A protocol to obtaining a raw starch degrading enzyme index (Ra/Ga) value is as follows:

-   1) The assays are performed at a temperature of 40° C. -   2) First, the pH profile of the enzyme is obtained on raw starch.     The profile is obtained from the plotting of the % activity versus     the pH. This optimum pH value is used in the assay. -   3) Any type of starch may be used, such as, wheat, corn, barley,     rice, etc. In an example, the raw starch used is corn starch. A 2%     solution of raw starch is used. Alternatively, to obtain the     gelatinized starch solution, a solution of raw starch is heated     above the gelatinization temperature for at least 60 minutes. In the     case of corn, the solution of raw starch is heated to 70° C. for at     least 60 minutes. -   4) The reaction solution contains the gelatinized starch (or raw     starch) and a buffer. The composition of the buffer used in the     assay depends on the pH optimum of the enzyme. The buffer     composition and concentration must be identical for both the raw and     gelatinized starch activity measurements. -   5) The enzyme concentration used in the assay must be identical for     both the raw and gelatinized starch activity measurements. -   6) The enzyme activity is measured by determination of the reducing     sugars in solution. Suitable methods are the following: The method     of Bernfield for determining reducing sugars using dinitrosalicylic     acid is described in Bernfield, 1955, Methods Enzymology 1:149-158     and the method for determining reducing sugars with     copper-bicinchoninate as described in Fox et al., 1991, Analytical     Biochemistry 195: 93-96 or in Waffenschmidt et al., 1987, Anal.     Biochem. 165: 337-340. Prior to the determination of reducing     sugars, the solutions are boiled for 3 minutes and centrifugated to     inactivate the enzyme. -   7) The time for incubation to measure the enzyme activities is 6     hours. -   8) The enzyme activity is expressed as the number reducing sugars     produced per hour and per mg of pure active enzyme. -   9) The activity on gelatinized starch is measured by measuring the     release of glucose produced by the enzyme on a 2% gelatinized (e.g.,     corn) starch reaction mixture and the activity on raw starch is     measured by measuring the release of glucose produced by the enzyme     on a 2% raw (e.g., corn) starch reaction mixture. The activity is     measured by the release of reducing sugars produced in 4 mol per     hour per mg of pure active enzyme.

EXAMPLES Example 1

Stability of Alpha-Amylase Variants

The stability of a reference alpha-amylase (Bacillus stearothermophilus alpha-amylase with the mutations I181*+G182*+N193F truncated to 491 amino acids (using SEQ ID NO: 1 herein for numbering) and alpha-amylase variants thereof was determined by incubating the reference alpha-amylase and variants at pH 4.5 and 5.5 and temperatures of 75° C. and 85° C. with 0.12 mM CaCl₂ followed by residual activity determination using the EnzChek® substrate (EnzChek® Ultra Amylase assay kit, E33651, Molecular Probes).

Purified enzyme samples were diluted to working concentrations of 0.5 and 1 or 5 and 10 ppm (micrograms/ml) in enzyme dilution buffer (10 mM acetate, 0.01% Triton X100, 0.12 mM CaCl₂, pH 5.0). Twenty microliters enzyme sample was transferred to 48-well PCR MTP and 180 microliters stability buffer (150 mM acetate, 150 mM MES, 0.01% Triton X100, 0.12 mM CaCl₂, pH 4.5 or 5.5) was added to each well and mixed. The assay was performed using two concentrations of enzyme in duplicates. Before incubation at 75° C. or 85° C., 20 microliters was withdrawn and stored on ice as control samples. Incubation was performed in a PCR machine at 75° C. and 85° C. After incubation samples were diluted to 15 ng/mL in residual activity buffer (100 mM Acetate, 0.01% Triton X100, 0.12 mM CaCl₂, pH 5.5) and 25 microliters diluted enzyme was transferred to black 384-MTP. Residual activity was determined using the EnzChek substrate by adding 25 microliters substrate solution (100 micrograms/ml) to each well. Fluorescence was determined every minute for 15 minutes using excitation filter at 485-P nm and emission filter at 555 nm (fluorescence reader is Polarstar, BMG). The residual activity was normalized to control samples for each setup.

Assuming logarithmic decay half life time (T½ (min)) was calculated using the equation: T½=T(min)*LN(0.5)/LN(% RA/100), where T is assay incubation time in minutes, and % RA is % residual activity determined in assay.

Using this assay setup the half life time was determined for the reference alpha-amylase and variant thereof as shown in Table 1.

TABLE 1 T½ (min) T½ (min) (pH 4.5, 85° C., T½ (min) (pH 4.5, 75° C., 0.12 mM (pH 5.5, 85° C., Mutations 0.12 mM CaCl₂) CaCl₂) 0.12 mM CaCl₂) Reference Alpha-Amylase A 21 4 111 Reference Alpha-Amylase A with the 32 6 301 substitution V59A Reference Alpha-Amylase A with the 28 5 230 substitution V59E Reference Alpha-Amylase A with the 28 5 210 substitution V59I Reference Alpha-Amylase A with the 30 6 250 substitution V59Q Reference Alpha-Amylase A with the 149 22 ND substitutions V59A + Q89R + G112D + E129V + K177L + R179E + K220P + N224L + Q254S Reference Alpha-Amylase A with the >180 28 ND substitutions V59A + Q89R + E129V + K177L + R179E + H208Y + K220P + N224L + Q254S Reference Alpha-Amylase A with the 112 16 ND substitutions V59A + Q89R + E129V + K177L + R179E + K220P + N224L + Q254S + D269E + D281N Reference Alpha-Amylase A with the 168 21 ND substitutions V59A + Q89R + E129V + K177L + R179E + K220P + N224L + Q254S + I270L Reference Alpha-Amylase A with the >180 24 ND substitutions V59A + Q89R + E129V + K177L + R179E + K220P + N224L + Q254S + H274K Reference Alpha-Amylase A with the 91 15 ND substitutions V59A + Q89R + E129V + K177L + R179E + K220P + N224L + Q254S + Y276F Reference Alpha-Amylase A with the 141 41 ND substitutions V59A + E129V + R157Y + K177L + R179E + K220P + N224L + S242Q + Q254S Reference Alpha-Amylase A with the >180 62 ND substitutions V59A + E129V + K177L + R179E + H208Y + K220P + N224L + S242Q + Q254S Reference Alpha-Amylase A with the >180 49 >480 substitutions V59A + E129V + K177L + R179E + K220P + N224L + S242Q + Q254S Reference Alpha-Amylase A with the >180 53 ND substitutions V59A + E129V + K177L + R179E + K220P + N224L + S242Q + Q254S + H274K Reference Alpha-Amylase A with the >180 57 ND substitutions V59A + E129V + K177L + R179E + K220P + N224L + S242Q + Q254S + Y276F Reference Alpha-Amylase A with the >180 37 ND substitutions V59A + E129V + K177L + R179E + K220P + N224L + S242Q + Q254S + D281N Reference Alpha-Amylase A with the >180 51 ND substitutions V59A + E129V + K177L + R179E + K220P + N224L + S242Q + Q254S + M284T Reference Alpha-Amylase A with the >180 45 ND substitutions V59A + E129V + K177L + R179E + K220P + N224L + S242Q + Q254S + G416V Reference Alpha-Amylase A with the 143 21 >480 substitutions V59A + E129V + K177L + R179E + K220P + N224L + Q254S Reference Alpha-Amylase A with the >180 22 ND substitutions V59A + E129V + K177L + R179E + K220P + N224L + Q254S + M284T Reference Alpha-Amylase A with the >180 38 ND substitutions A91L + M96I + E129V + K177L + R179E + K220P + N224L + S242Q + Q254S Reference Alpha-Amylase A with the 57 11 402 substitutions E129V + K177L + R179E Reference Alpha-Amylase A with the 174 44 >480 substitutions E129V + K177L + R179E + K220P + N224L + S242Q + Q254S Reference Alpha-Amylase A with the >180 49 >480 substitutions E129V + K177L + R179E + K220P + N224L + S242Q + Q254S + Y276F + L427M Reference Alpha-Amylase A with the >180 49 >480 substitutions E129V + K177L + R179E + K220P + N224L + S242Q + Q254S + M284T Reference Alpha-Amylase A with the 177 36 >480 substitutions E129V + K177L + R179E + K220P + N224L + S242Q + Q254S + N376* + I377* Reference Alpha-Amylase A with the 94 13 >480 substitutions E129V + K177L + R179E + K220P + N224L + Q254S Reference Alpha-Amylase A with the 129 24 >480 substitutions E129V + K177L + R179E + K220P + N224L + Q254S + M284T Reference Alpha-Amylase A with the 148 30 >480 substitutions E129V + K177L + R179E + S242Q Reference Alpha-Amylase A with the 78 9 >480 substitutions E129V + K177L + R179V Reference Alpha-Amylase A with the 178 31 >480 substitutions E129V + K177L + R179V + K220P + N224L + S242Q + Q254S Reference Alpha-Amylase A with the 66 17 >480 substitutions K220P + N224L + S242Q + Q254S Reference Alpha-Amylase A with the 30 6 159 substitutions K220P + N224L + Q254S Reference Alpha-Amylase A with the 35 7 278 substitution M284T Reference Alpha-Amylase A with the 59 13 ND substitutions M284V ND not determined

The results demonstrate that the alpha-amylase variants have a significantly greater half-life and stability than the reference alpha-amylase.

Example 2

Preparation of Protease Variants and Test of Thermostability

Chemicals used were commercial products of at least reagent grade.

Strains and Plasmids:

E. coli DH12S (available from Gibco BRL) was used for yeast plasmid rescue. pJTP000 is a S. cerevisiae and E. coli shuttle vector under the control of TPI promoter, constructed from pJC039 described in WO 01/92502, in which the Thermoascus aurantiacus M35 protease gene (WO 03/048353) has been inserted.

Saccharomyces cerevisiae YNG318 competent cells: MATa Dpep4[cir+] ura3-52, leu2-D2, his 4-539 was used for protease variants expression. It is described in J. Biol. Chem. 272(15): 9720-9727 (1997).

Media and Substrates

10× Basal Solution:

Yeast nitrogen base w/o amino acids (DIFCO) 66.8 g/L, succinate 100 g/I, NaOH 60 g/I.

Sc-Glucose:

20% glucose (i.e., a final concentration of 2%=2 g/100 mL)) 100 mL/L, 5% threonine 4 mL/L, 1% tryptophan10 ml/l, 20% casamino acids 25 ml/l, 10× basal solution 100 ml/l. The solution is sterilized using a filter of a pore size of 0.20 micrometer. Agar (2%) and H₂O (approx. 761 mL) is autoclaved together, and the separately sterilized SC-glucose solution is added to the agar solution.

YPD:

Bacto peptone 20 g/l, yeast extract 10 g/L, 20% glucose 100 mL/L.

YPD+Zn:

YPD+0.25 mM ZnSO₄.

PEG/LiAc solution:

40% PEG4000 50 ml, 5 M Lithium Acetate 1 mL.

96 Well Zein Micro Titre Plate:

Each well contains 200 microL of 0.05-0.1% of zein (Sigma), 0.25 mM ZnSO₄ and 1% of agar in 20 mM sodium acetate buffer, pH 4.5.

DNA Manipulations

Unless otherwise stated, DNA manipulations and transformations were performed using standard methods of molecular biology as described in Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab. Cold Spring Harbor, N.Y.; Ausubel, F. M. et al. (eds.) “Current protocols in Molecular Biology”, John Wiley and Sons, 1995; Harwood, C. R. and Cutting, S. M. (Eds.).

Yeast Transformation

Yeast transformation was performed using the lithium acetate method. 0.5 microL of vector (digested by restriction endnucleases) and 1 microL of PCR fragments is mixed. The DNA mixture, 100 microL of YNG318 competent cells, and 10 microL of YEAST MAKER carrier DNA (Clontech) is added to a 12 mL polypropylene tube (Falcon 2059). Add 0.6 mL PEG/LiAc solution and mix gently. Incubate for 30 min at 30° C., and 200 rpm followed by 30 min at 42° C. (heat shock). Transfer to an eppendorf tube and centrifuge for 5 sec. Remove the supernatant and resolve in 3 mL of YPD. Incubate the cell suspension for 45 min at 200 rpm at 30° C. Pour the suspension to SC-glucose plates and incubate 30° C. for 3 days to grow colonies. Yeast total DNA are extracted by Zymoprep Yeast Plasmid Miniprep Kit (ZYMO research).

DNA Sequencing

E. coli transformation for DNA sequencing was carried out by electroporation (BIO-RAD Gene Pulser). DNA Plasmids were prepared by alkaline method (Molecular Cloning, Cold Spring Harbor) or with the Qiagen® Plasmid Kit. DNA fragments were recovered from agarose gel by the Qiagen gel extraction Kit. PCR was performed using a PTC-200 DNA Engine. The ABI PRISM™ 310 Genetic Analyzer was used for determination of all DNA sequences.

Construction of Protease Expression Vector

The Themoascus M35 protease gene was amplified with the primer pair Prot F (SEQ ID NO: 4) and Prot R (SEQ ID NO: 5). The resulting PCR fragments were introduced into S. cerevisiae YNG318 together with the pJC039 vector (described in WO 2001/92502) digested with restriction enzymes to remove the Humicola insolens cutinase gene.

The Plasmid in yeast clones on SC-glucose plates was recovered to confirm the internal sequence and termed as pJTP001.

Construction of Yeast Library and Site-Directed Variants

Library in yeast and site-directed variants were constructed by SOE PCR method (Splicing by Overlap Extension, see “PCR: A practical approach”, p. 207-209, Oxford University press, eds. McPherson, Quirke, Taylor), followed by yeast in vivo recombination.

General Primers for Amplification and Sequencing

The primers AM34 (SEQ ID NO: 6) and AM35 (SEQ ID NO:7) were used to make DNA fragments containing any mutated fragments by the SOE method together with degenerated primers (AM34+Reverse primer and AM35+forward primer) or just to amplify a whole protease gene (AM34+AM35).

PCR reaction system: Conditions: 48.5 microL H₂O 1 94° C. 2 min 2 beads puRe Taq Ready-To-Go PCR (Amersham 2 94° C. 30 sec Biosciences) 0.5 microL × 2 100 pmole/microL of primers 3 55° C. 30 sec 0.5 microL template DNA 4 72° C. 90 sec 2-4 25 cycles 5 72° C. 10 min

DNA fragments were recovered from agarose gel by the Qiagen gel extraction Kit. The resulting purified fragments were mixed with the vector digest. The mixed solution was introduced into Saccharomyces cerevisiae to construct libraries or site-directed variants by in vivo recombination.

Relative Activity Assay

Yeast clones on SC-glucose were inoculated to a well of a 96-well micro titre plate containing YPD+Zn medium and cultivated at 28° C. for 3 days. The culture supernatants were applied to a 96-well zein micro titer plate and incubated at at least 2 temperatures (ex., 70° C. and 80° C.) for more than 4 hours or overnight. The turbidity of zein in the plate was measured as A630 and the relative activity (higher/lower temperatures) was determined as an indicator of thermoactivity improvement. The clones with higher relative activity than the parental variant were selected and the sequence was determined.

Remaining Activity Assay

Yeast clones on SC-glucose were inoculated to a well of a 96-well micro titre plate and cultivated at 28° C. for 3 days. Protease activity was measured at 65° C. using azo-casein (Megazyme) after incubating the culture supernatant in 20 mM sodium acetate buffer, pH 4.5, for 10 min at a certain temperature (80° C. or 84° C. with 4° C. as a reference) to determine the remaining activity. The clones with higher remaining activity than the parental variant were selected and the sequence was determined.

Azo-Casein Assay

20 microL of samples were mixed with 150 microL of substrate solution (4 mL of 12.5% azo-casein in ethanol in 96 mL of 20 mM sodium acetate, pH 4.5, containing 0.01% triton-100 and 0.25 mM ZnSO₄) and incubated for 4 hours or longer.

After adding 20 microL/well of 100% trichloroacetic acid (TCA) solution, the plate was centrifuge and 100 microL of supernatants were pipette out to measure A440.

Expression of Protease Variants in Aspergillus oryzae

The constructs comprising the protease variant genes were used to construct expression vectors for Aspergillus. The Aspergillus expression vectors consist of an expression cassette based on the Aspergillus niger neutral amylase II promoter fused to the Aspergillus nidulans triose phosphate isomerase non translated leader sequence (Pna2/tpi) and the Aspergillus niger amyloglycosidase terminator (Tamg). Also present on the plasmid was the Aspergillus selective marker amdS from Aspergillus nidulans enabling growth on acetamide as sole nitrogen source. The expression plasmids for protease variants were transformed into Aspergillus as described in Lassen et al., 2001, Appl. Environ. Microbiol. 67: 4701-4707. For each of the constructs 10-20 strains were isolated, purified and cultivated in shake flasks.

Purification of Expressed Variants

Adjust pH of the 0.22 micro m filtered fermentation sample to 4.0.

Put the sample on an ice bath with magnetic stirring. Add (NH₄)₂SO₄ in small aliquots (corresponding to approx. 2.0-2.2 M (NH₄)₂SO₄ not taking the volume increase into account when adding the compound).

After the final addition of (NH₄)₂SO₄, incubate the sample on the ice bath with gentle magnetic stirring for min. 45 min.

Centrifugation: Hitachi himac CR20G High-Speed Refrigerated Centrifuge equipped with R20A2 rotor head, 5° C., 20,000 rpm, 30 min.

Dissolve the formed precipitate in 200 mL 50 mM Na-acetate pH 4.0.

Filter the sample by vacuum suction using a 0.22 micro m PES PLUS membrane (IWAKI).

Desalt/buffer-exchange the sample to 50 mM Na-acetate pH 4.0 using ultrafiltration (Vivacell 250 from Vivascience equipped with 5 kDa MWCO PES membrane) overnight in a cold room. Dilute the retentate sample to 200 ml using 50 mM Na-acetate pH 4.0. The conductivity of sample is preferably less than 5 mS/cm.

Load the sample onto a cation-exchange column equilibrated with 50 mM Na-acetate pH 4.0. Wash unbound sample out of the column using 3 column volumes of binding buffer (50 mM Na-acetate pH 4.0), and elute the sample using a linear gradient, 0-100% elution buffer (50 mM Na-acetate+1 M NaCl pH 4.0) in 10 column volumes.

The collected fractions are assayed by an endo-protease assay (cf. below) followed by standard SDS-PAGE (reducing conditions) on selected fractions. Fractions are pooled based on the endo-protease assay and SDS-PAGE.

Endo-Protease Assay

Protazyme OL tablet/5 ml 250 mM Na-acetate pH 5.0 is dissolved by magnetic stirring (substrate: endo-protease Protazyme AK tablet from Megazyme—cat. #PRAK 11/08).

With stirring, 250 microL of substrate solution is transferred to a 1.5 mL Eppendorf tube.

25 microL of sample is added to each tube (blank is sample buffer).

The tubes are incubated on a Thermomixer with shaking (1000 rpm) at 50° C. for 15 minutes.

250 microL of 1 M NaOH is added to each tube, followed by vortexing.

Centrifugation for 3 min. at 16,100×G and 25° C.

200 microL of the supernatant is transferred to a MTP, and the absorbance at 590 nm is recorded.

TABLE 2 Relative Activity of protease variants. Numbering of substitution(s) starts from N-terminal of the mature peptide in amino acids 1 to 177 of SEQ ID NO: 3. Remaining Activity Variant Substitution(s) and/or deletion(s) 80° C. 84° JTP082 ΔS5/D79L/S87P/A112P/D142L 53% JTP091 D79L/S87P/A112P/T124V/D142L 43% JTP092 ΔS5/N26R/D79L/S87P/A112P/D142L 60% JTP095 N26R/T46R/D79L/S87P/A112P/D142L 62% JTP096 T46R/D79L/S87P/T116V/D142L 67% JTP099 D79L/P81R/S87P/A112P/D142L 80% JTP101 A27K/D79L/S87P/A112P/T124V/D142L 81% JTP116 D79L/Y82F/S87P/A112P/T124V/D142L 59% JTP117 D79L/Y82F/S87P/A112P/T124V/D142L 94% JTP127 D79L/S87P/A112P/T124V/A126V/D142L 53%

TABLE 3 Relative Activity of protease variants. Numbering of substitution(s) starts from N- terminal of the mature peptide in amino acids 1 to 177 of SEQ ID NO: 3. Relative Activity Variant Substitutions 80° C./70° C. 85° C./70° C. JTP050 D79L S87P A112P D142L 23% 9% JTP134 D79L Y82F S87P A112P D142L 40% JTP135 S38T D79L S87P A112P A126V D142L 62% JTP136 D79L Y82F S87P A112P A126V D142L 59% JTP137 A27K D79L S87P A112P A126V D142L 54% JTP145 S49P D79L S87P A112P D142L 59% JTP146 S50P D79L S87P A112P D142L 63% JTP148 D79L S87P D104P A112P D142L 64% JTP161 D79L Y82F S87G A112P D142L 30% 12% JTP180 S70V D79L Y82F S87G Y97W A112P D142L 52% JTP181 D79L Y82F S87G Y97W D104P A112P D142L 45% JTP187 S70V D79L Y82F S87G A112P D142L 45% JTP188 D79L Y82F S87G D104P A112P D142L 43% JTP189 D79L Y82F S87G A112P A126V D142L 46% JTP193 Y82F S87G S70V D79L D104P A112P D142L 15% JTP194 Y82F S87G D79L D104P A112P A126V D142L 22% JTP196 A27K D79L Y82F S87G D104P A112P A126V 18% D142L

TABLE 4 Relative Activity of protease variants. Numbering of substitution(s) starts from N-terminal of the mature peptide in amino acids 1 to 177 of SEQ ID NO: 3. Relative Activity Variant Substitutions 80° C./70° C. JTP196 A27K D79L Y82F S87G D104P A112P 55% A126V D142L JTP210 A27K Y82F S87G D104P A112P A126V 36% D142L JTP211 A27K D79L Y82F D104P A112P A126V 44% D142L JTP213 A27K Y82F D104P A112P A126V D142L 37%

Example 3

Temperature Profile of Selected Protease Variants Using Purified Enzymes

Selected protease variants showing good thermostability were purified and the purified enzymes were used in a zein-BCA assay as described below. The remaining protease activity was determined at 60° C. after incubation of the enzyme at elevated temperatures as indicated for 60 min.

Zein-BCA Assay:

Zein-BCA assay was performed to detect soluble protein quantification released from zein by variant proteases at various temperatures.

Protocol:

Mix 10 microL of 10 micro g/mL enzyme solutions and 100 microL of 0.025% zein solution in a micro titer plate (MTP).

Incubate at various temperatures for 60 min.

Add 10 microL of 100% trichloroacetic acid (TCA) solution.

Centrifuge MTP at 3500 rpm for 5 min.

Take out 15 microL to a new MTP containing 100 microL of BCA assay solution (Pierce Cat#:23225, BCA Protein Assay Kit).

Incubate for 30 min. at 60° C.

Measure A562.

The results are shown in Table 5. All of the tested protease variants showed an improved thermostability as compared to the wild type (WT) protease.

TABLE 5 Zein-BCA assay Sample incubated 60 min at indicated temperatures (° C.) (micro g/mL Bovine serum albumin equivalent peptide released) WT/Variant 60° C. 70° C.  75° C.  80° C. 85° C. 90° C. 95° C. WT 94 103 107 93 58 38 JTP050 (D79L + S87P + 86 101 107 107 104 63 36 A112P + D142L) JTP077 (A27K + D79L + 82 94 104 105 99 56 31 S87P + A112P + D142L) JTP188 (D79L + Y82F + 71 83 86 93 100 75 53 S87G + D104P + A112P + D142L) JTP196 (A27K + D79L + 87 99 103 106 117 90 38 Y82F + S87G + D104P + A112P + A126V + D142L)

Example 4

Characterization of Penicillium oxalicum glucoamylase

The Penicillium oxalicum glucoamylase is disclosed in SEQ ID NO: 9 herein.

Substrate. Substrate: 1% soluble starch (Sigma S-9765) in deionized water

Reaction buffer: 0.1 M Acetate buffer at pH 5.3

Glucose concentration determination kit: Wako glucose assay kit (LabAssay glucose, WAKO, Cat#298-65701).

Reaction condition. 20 microL soluble starch and 50 microL acetate buffer at pH5.3 were mixed. 30 microL enzyme solution (50 micro g enzyme protein/ml) was added to a final volume of 100 microL followed by incubation at 37° C. for 15 min.

The glucose concentration was determined by Wako kits.

All the work carried out in parallel.

Temperature Optimum.

To assess the temperature optimum of the Penicillium oxalicum glucoamylase the “Reaction condition”-assay described above was performed at 20, 30, 40, 50, 60, 70, 80, 85, 90 and 95° C. The results are shown in Table 6.

TABLE 6 Temperature optimum Temperature (° C.) 20 30 40 50 60 70 80 85 90 95 Relative 63.6 71.7 86.4 99.4 94.6 100.0 92.9 92.5 82.7 82.8 activity (%)

From the results it can be seen that the optimal temperature for Penicillium oxalicum glucoamylase at the given conditions is between 50° C. and 70° C. and the glucoamylase maintains more than 80% activity at 95° C.

Heat Stability.

To assess the heat stability of the Penicillium oxalicum glucoamylase the Reaction condition assay was modified in that the the enzyme solution and acetate buffer was preincubated for 15 min at 20, 30, 40, 50, 60, 70, 75, 80, 85, 90 and 95° C. Following the incubation 20 microL of starch was added to the solution and the assay was performed as described above.

The results are shown in Table 7.

TABLE 7 Heat stability Temperature (° C.) 20 30 40 50 60 70 80 85 90 95 Relative 91.0 92.9 88.1 100.0 96.9 86.0 34.8 36.0 34.2 34.8 activity (%)

From the results it can be seen that Penicillium oxalicum glucoamylase is stable up to 70° C. after preincubation for 15 min in that it maintains more than 80% activity.

pH Optimum.

To assess the pH optimum of the Penicillium oxalicum glucoamylase the Reaction condition assay described above was performed at pH 2.0, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0 7.0, 8.0, 9.0, 10.0 and 11.0. Instead of using the acetate buffer described in the Reaction condition assay the following buffer was used 100 mM Succinic acid, HEPES, CHES, CAPSO, 1 mM CaCl₂, 150 mM KCl, 0.01% Triton X-100, pH adjusted to 2.0, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0 7.0, 8.0, 9.0, 10.0 or 11.0 with HCl or NaOH.

The results are shown in Table 8.

TABLE 8 pH optimum pH 2.0 3.0 3.5 4.0 4.5 5.0 6.0 7.0 8.0 9.0 10.0 11.0 Relative 71.4 78.6 77.0 91.2 84.2 100.0 55.5 66.7 30.9 17.8 15.9 16.1 activity (%)

From the results it can be seen that Penicillium oxalicum glucoamylase at the given conditions has the highest activity at pH 5.0. The Penicillium oxalicum glucoamylase is active in a broad pH range in the it maintains more than 50% activity from pH 2 to 7.

pH Stability.

To assess the heat stability of the Penicillium oxalicum glucoamylase the Reaction condition assay was modified in that the enzyme solution (50 micro g/mL) was preincubated for 20 hours in buffers with pH 2.0, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0 7.0, 8.0, 9.0, 10.0 and 11.0 using the buffers described under pH optimum. After preincubation, 20 microL soluble starch to a final volume of 100 microL was added to the solution and the assay was performed as described above.

The results are shown in Table 9.

TABLE 9 pH stability pH 2.0 3.0 3.5 4.0 4.5 5.0 6.0 7.0 8.0 9.0 10.0 11.0 Relative 17.4 98.0 98.0 103.2 100.0 93.4 71.2 90.7 58.7 17.4 17.0 17.2 activity (%)

From the results it can be seen that Penicillium oxalicum glucoamylase, is stable from pH 3 to pH 7 after preincubation for 20 hours and it decreases its activity at pH 8.

Example 5

Improved Ethanol Production Process with Liquefaction at 75° C., pH 4.80

Ground corn and backset were used for this study. The dry solids (% DS) contents of the ground corn and backset were measured to be 86.78 and 7.93%, respectively, by oven drying at 105° C. for 3 hours.

Mash Preparation.

Nine different corn slurries were prepared for liquefaction. Into 5×200 g lab-o-mat canisters (Mathis, Inc.) were added 35.29 g of ground corn, 37.71 g of tap water, and 30.00 g of backset. The backset ratio was set to 30% for all slurries. Following the addition of these components, the slurries were pH-adjusted to 4.80 using 40% H₂SO₄ if needed. Stock solutions of all concentrated enzymes were prepared using deionized water. Aliquots of all enzymes were added to each lab-o-mat canister to reach the final concentrations specified in Table 10. Finally, deionized water was added to the slurries to ensure that the starting % DS's for all slurries were identical.

TABLE 10 Amylase Dose Units RSH Amylase Dose Units AMG Dose Units pH Temp. Glucoamylase Dose Units 1 BAA 1407 1.4 ug/gDS ug/gDS 4.8 75 AMG SPU 0.5 AGU/gDS 2 BAA1407 1.4 ug/gDS RSH AA 96 3 ug/gDS 4.8 75 AMG SPU 0.5 AGU/gDS 3 BAA 1407 1.4 ug/gDS RSH AA 96 10 ug/gDS 4.8 75 AMG SPU 0.5 AGU/gDS 4 BAA 1407 1.4 ug/gDS RSH AA 96 25 ug/gDS 4.8 75 AMG SPU 0.5 AGU/gDS 5 BAA 1407 1.4 ug/gDS RSH AA 101 3 ug/gDS 4.8 75 AMG SPU 0.5 AGU/gDS 6 BAA 1407 1.4 ug/gDS RSH AA 101 10 ug/gDS 4.8 75 AMG SPU 0.5 AGU/gDS 7 BAA 1407 1.4 ug/gDS RSH AA 101 25 ug/gDS 4.8 75 AMG SPU 0.5 AGU/gDS 8 BAA 1407 1.4 ug/gDS RSH AA 96 25 ug/gDS AMG 001 10 ug/gDS 4.8 75 AMG SPU 0.5 AGU/gDS 9 BAA 1407 1.4 ug/gDS RSH AA 101 25 ug/gDS AMG 001 10 ug/gDS 4.8 75 AMG SPU 0.5 AGU/gDS

All canisters were then closed. They were placed into the lab-o-mat and the following program was used for liquefaction (Table 11):

TABLE 11 Liquefaction: Ramp 5 deg/min rpm = 30 Lab-o-mat Ramp time 17 min Liq temp 75° C. Liq time 113 min

At the end of the program described in Table 11, all canisters were removed from the lab-o-mat and cooled immediately in an ice bath.

Fermentation Setup.

After complete cooling, urea and penicillin were added to all mashes to reach final concentrations of 750 and 3 ppm, respectively. Mashes were adjusted to pH 5.0 using 40% H₂SO₄ or 50% NaOH. The final % dry solids of the mashes were measured and recorded as 32.44%.

Approximately 5 g of each mash were transferred into preweighed 15 mL plastic Falcon centrifuge tubes for fermentation. A small hole was drilled into the lid of each tube to allow for CO₂ release during fermentation. Five replicate fermentations were prepared for each treatment. Following mash transfer, all tubes were reweighed to obtain the initial sample weights. Into each tube was then added 100 microL of rehydrated Red Star Ethanol Red yeast (rehydrated by weighing 5.5 g of dry yeast into a 150 mL Erlenmeyer flask, adding 100 mL of tap water, and stirring in a 32° C. water bath for 30 minutes), an aliquot of diluted AMG SPU glucoamylase (diluted in deionized water) needed to reach a starting concentration of 0.50 AGU/g DS. An appropriate amount of deionized water was finally added to each tube such that the total volume of liquid added to each tube relative to the sample weight was the same. All tubes were then reweighed and then placed into a preheated water bath set at 32° C. Fermentation was allowed to progress for a total of 54 hours. Tubes were vigorously vortexed after approximately 7 hours and then reweighed twice per day for the remaining fermentation time. The grams of ethanol produced per gram of dry solids in each tube was calculated from the weight loss data according to the following equation:

${g\mspace{14mu}{ethanol}\text{/}g\mspace{14mu}{DS}} = \frac{\begin{matrix} {g\mspace{14mu}{CO}_{2}\mspace{14mu}{weight}\mspace{14mu}{loss} \times \frac{1\mspace{14mu}{mol}\mspace{20mu}{CO}_{2}}{44.0098\mspace{14mu} g\mspace{14mu}{CO}_{2}} \times} \\ {\frac{1\mspace{14mu}{mol}\mspace{14mu}{ethanol}}{1\mspace{14mu}{mol}\mspace{14mu}{CO}_{2}} \times \frac{46.094\mspace{14mu} g\mspace{14mu}{ethanol}}{1\mspace{14mu}{mol}\mspace{14mu}{ethanol}}} \end{matrix}}{g\mspace{14mu}{corn}\mspace{14mu}{in}\mspace{14mu}{tube} \times \%\mspace{14mu}{DS}\mspace{14mu}{of}\mspace{14mu}{corn}}$

Three of the replicate tubes were pulled after 54 hours of fermentation for HPLC analysis. Pulled samples were treated with 50 microL of 40% H₂SO₄ to stop fermentation and vortexed thoroughly. The samples were then centrifuged at 1570×g for 10 minutes and then filtered into HPLC vials through 0.45 micro m syringe filters. HPLC analysis was finally conducted on the samples to quantify the amounts of DP4+, DP3, DP2, glucose, fructose, lactic and acetic acids, glycerol, and ethanol.

Results

FIG. 1 shows the averaged HPLC results obtained for the treatments after 54 hours of fermentation. A significant increase in ethanol production was measured when RSH AA 96, RSH AA 101, and AMG 001 glucoamylase were added into liquefaction on top of BAA 1407 alpha amylase.

The Present Invention is Described in the Following Paragraphs:

Paragraph 1. A process for producing fermentation products from starch-containing material comprising the steps of:

i) liquefying the starch-containing material at a temperature in the range from 60-80° C. using:

-   -   a bacterial alpha-amylase;     -   a raw starch hydrolyzing alpha-amylase;     -   a carbohydrate-source generating enzyme having a heat stability         at 70° C., pH 5.3, of at least 70%;

ii) saccharifying using a carbohydrate-source generating enzyme;

iii) fermenting using a fermenting organism.

Paragraph 2. The process of paragraph 1, further comprises, prior to the liquefaction step i), the steps of:

a) reducing the particle size of the starch-containing material, preferably by dry milling;

b) forming a slurry comprising the starch-containing material and water.

Paragraph 3. The process of paragraph 1 or 2, wherein at least 50%, preferably at least 70%, more preferably at least 80%, especially at least 90% of the starch-containing material fit through a sieve with #6 screen.

Paragraph 4. The process of any of paragraphs 1-3, wherein the pH during liquefaction step i) is in the range from 4-6, preferably from 4.5-5.0 or between 4.5-4.8 or between 5.0 and 6.0.

Paragraph 5. The process of any of paragraphs 1-4, wherein the temperature during liquefaction is in the range between from 70-80° C. such as 75-80° C., preferably around 75° C.

Paragraph 6. The process of any of paragraphs 1-5, wherein liquefaction is carried out for 0.1-10 hours, such as 1-3 hours, such as around 1.5 hours.

Paragraph 7. The process of paragraph 6, wherein a jet-cooking step is carried out after liquefaction in step i), such as wherein the jet-cooking is carried out at a temperature between 110-145° C., preferably 120-140° C., such as 125-135° C., preferably around 130° C. for about 1-15 minutes, preferably for about 3-10 minutes, especially around about 5 minutes. Paragraph 8. The process of any of paragraphs 1-7, wherein saccharification and fermentation is carried out sequentially or simultaneously. Paragraph 9. The process of any of paragraphs 1-8, wherein saccharification is carried out at a temperature from 20-75° C., preferably from 40-70° C., such as around 60° C., and at a pH between 4 and 5, such as around pH 4.5. Paragraph 10. The process of any of paragraphs 1-9, wherein fermentation or simultaneous saccharification and fermentation (SSF) is carried out at a temperature from 25° C. to 40° C., such as from 28° C. to 35° C., such as from 30° C. to 34° C., preferably around about 32° C., wherein fermentation is ongoing for 6 to 120 hours, in particular 24 to 96 hours. Paragraph 11. The process of any of paragraphs 1-10, wherein the fermentation product is an alcohol, preferably ethanol, especially fuel ethanol, potable ethanol and/or industrial ethanol. Paragraph 12. The process of any of paragraphs 1-11, wherein the fermentation product is recovered after fermentation, such as by distillation. Paragraph 13. The process of any of paragraphs 1-12, wherein the starch-containing starting material is whole grains. Paragraph 14. The process of any of paragraphs 1-13, wherein the starch-containing material is derived from corn, wheat, barley, rye, milo, sago, cassava, manioc, tapioca, sorghum, rice or potatoes. Paragraph 15. The process of any of paragraphs 1-14, wherein the fermenting organism is yeast, preferably a strain of Saccharomyces. Paragraph 16. The process of any of paragraphs 1-15, wherein the fermenting organism is a strain of Saccharomyces cerevisae. Paragraph 17. The process of paragraphs 1-16, wherein the bacterial alpha-amylase is derived from a strain of Bacillus (also referred to as Geobacillus). Paragraph 18. The process of paragraph 17, wherein the bacterial alpha-amylase is of the genus Bacillus or Geobacillus, such as a strain of Bacillus stearothermophilus, in particular a variant of a Bacillus stearothermophilus alpha-amylase or Geobacillus stearothermophilus, such as the one shown in SEQ ID NO: 3 in WO 99/019467 or SEQ ID NO: 1 herein, in particular the Bacillus stearothermophilus alpha-amylase is truncated, preferably to have 491 amino acids. Paragraph 19. The process of any of paragraphs 1-18, wherein the bacterial alpha-amylase has at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity to the mature part of the polypeptide of SEQ ID NO: 3 disclosed in WO 99/019467 or SEQ ID NO: 1 herein. Paragraph 20. The process of any of paragraphs 17-19, wherein the bacterial alpha-amylase has a T½ (min) at pH 4.5, 75° C., 0.12 mM CaCl₂) of at least 20, such as at least 25, such as at least 30, such as at least 40, such as at least 50, such as at least 60, such as at least 70, such as at least 80, such as at least 90, such as at least 100, such as at least 110, such as at least 120, such as at least 130, such as at least 140, such as at least 150, such as at least 160, such as at least 170, such as at least 180 such as between 20-300, such as between 50-300, such as between 60-300, such as between 70-300, such as between 80-300, such as between 90-300, such as between 100-300, such as between 120-300, such as between 140-300 such as between 160-300, such as between 180-300. Paragraph 21. The process of any of paragraphs 17-20, wherein the bacterial alpha-amylase is derived from Bacillus stearothermophilus alpha-amylase truncated to have 491 amino acids with mutations selected from the group consisting of:

-   -   V59A+Q89R+E129V+K177L+R179E+I181*+G182*+N193F+H208Y+K220P+N224L+Q254S;     -   E129V+K177L+R179E+I181*+G182*+N193F; and     -   E129V+K177L+R179E+I181*+G182*+N193F+K220P+N224L+S242Q+Q254S.         Paragraph 22. The process of any of paragraphs 17-21, wherein         the Bacillus stearothermophilus or Geobacillus         stearothermophilus alpha-amylase is a variant with the following         mutations: I181*+G182*, preferably I181*+G182*+N193F using SEQ         ID NO: 3 in WO 99/019467 or SEQ ID NO: 1 herein for the         numbering.         Paragraph 23. The process of paragraph 1, wherein the bacterial         alpha-amylase is a chimeric alpha-amylase disclosed in         Richardson et al., 2002, The Journal of Biological Chemistry         277(29): 26501-26507, preferably the one referred to as BD5088         or shown as amino acids 1 to 435 of SEQ ID NO: 2 in WO         2007/134207.         Paragraph 24. The process of paragraphs 1-23, wherein the raw         starch hydrolyzing alpha-amylase is of fungal origin, preferably         a variant of Rhizomucor pusillus alpha-amylase with Aspergillus         niger glucoamylase linker and SBD and further one or more of the         following substitutions: G128D, D143N, K192R, such as         G128D+D143N or G128D+D143N+K192R (using SEQ ID NO: 14 herein for         the numbering).         Paragraph 25. The process of any of paragraphs 1-24, further         wherein the carbohydrate-source generating enzyme present and/or         added during liquefaction step i) is a glucoamylase.         Paragraph 26. The process of paragraph 25, wherein the         carbohydrate-source generating enzyme is a glucoamylase having a         heat stability at 70° C., pH 5.3, of at least 75%, preferably at         least 80%, preferably at least 85%.         Paragraph 27. The process of paragraph 25 or 26, wherein the         carbohydrate-generating enzyme is a glucoamylase having a         relative activity at pH 4.5 of at least 80%, preferably at least         85%, preferably at least 90%.         Paragraph 28. The process of any of paragraphs 25-27, wherein         the carbohydrate-generating enzyme is a glucoamylase having a pH         stability at pH 4.5 of at least at least 80%, at least 85%, at         least 90%, at least 95%, at least 100%.         Paragraph 29. The process of any of paragraphs 25-28, wherein         the carbohydrate-source generating enzyme is a glucoamylase,         preferably derived from a strain of the genus Penicillium,         especially a strain of Penicillium oxalicum disclosed as SEQ ID         NO: 2 in PCT/CN10/071753 published as WO 2011/127802 or SEQ ID         NO: 9 and 15 herein, or a variant thereof having a K79V         substitution (using SEQ ID NO: 15 herein for numbering).         Paragraph 30. The process of any of paragraphs 25-29, wherein         the glucoamylase has at least 80%, more preferably at least 85%,         more preferably at least 90%, more preferably at least 91%, more         preferably at least 92%, even more preferably at least 93%, most         preferably at least 94%, and even most preferably at least 95%,         such as even at least 96%, at least 97%, at least 98%, at least         99% or 100% identity to the mature polypeptide shown in SEQ ID         NO: 2 in PCT/CN10/071753 published as WO 2011/127802 or SEQ ID         NO: 9 herein.         Paragraph 31. The process of any of paragraphs 1-30, further         wherein a glucoamylase is present and/or added during         saccharification and/or fermentation.         Paragraph 32. The process of any of paragraphs 1-31, wherein the         glucoamylase present and/or added during saccharification and/or         fermentation is of fungal origin, preferably from a stain of         Aspergillus, preferably A. niger, A. awamori, or A. oryzae; or a         strain of Trichoderma, preferably T. reesei; or a strain of         Talaromyces, preferably T. emersonii, or a strain of Pycnoporus,         or a strain of Gloephyllum.         Paragraph 33. The process of any of paragraphs 1-32, further         wherein a protease is present or added during liquefaction.         Paragraph 34. The process of paragraph 33, wherein the protease         is of fungal or bacterial origin.         Paragraph 35. The process of paragraph 33 or 34, wherein the         protease has a thermostability value of more than 20% determined         as Relative Activity at 80° C./70° C.         Paragraph 36. The process of any of paragraphs 33-35, wherein         the protease has a thermostability value of more than 30%, more         than 40%, more than 50%, more than 60%, more than 70%, more than         80%, more than 90% determined as Relative Activity at 80° C./70°         C.         Paragraph 37. The process of any of paragraphs 33-36, wherein         the protease is a metallo protease.         Paragraph 38. The process of any of paragraphs 33-37, wherein         the protease is a variant of the metallo protease derived from a         strain of the genus Thermoascus, preferably a strain of         Thermoascus aurantiacus, especially Thermoascus aurantiacus         CGMCC No. 0670.         Paragraph 39. The process of any of paragraphs 33-38, wherein         the protease is a variant of the metallo protease disclosed as         the mature part of SEQ ID NO. 2 disclosed in WO 2003/048353 or         the mature part of SEQ ID NO: 1 in WO 2010/008841 or SEQ ID NO:         3 herein.         Paragraph 40. The process of any of paragraphs 33-39, wherein         the protease variant has at least 75% identity preferably at         least 80%, more preferably at least 85%, more preferably at         least 90%, more preferably at least 91%, more preferably at         least 92%, even more preferably at least 93%, most preferably at         least 94%, and even most preferably at least 95%, such as even         at least 96%, at least 97%, at least 98%, at least 99%, but less         than 100% identity to the mature part of the polypeptide of SEQ         ID NO: 2 disclosed in WO 2003/048353 or the mature part of SEQ         ID NO. 1 in WO 2010/008841 or SEQ ID NO: 3 herein.         Paragraph 41. The process of any of paragraphs 33-40, wherein         the protease is derived from a strain of Pyrococcus.         Paragraph 42. The process of any of paragraphs 33-41, wherein         the protease is derived from a strain of Pyrococcus furiosus.         Paragraph 43. The process of any of paragraphs 33-42 wherein the         protease is the one shown in SEQ ID NO: 1 in U.S. Pat. No.         6,258,726 or SEQ ID NO: 13 herein.         Paragraph 44. The process of any of paragraphs 33-43, wherein         the protease is one having at least 80%, such as at least 85%,         such as at least 90%, such as at least 95%, such as at least         96%, such as at least 97%, such as at least 98%, such as at         least 99% identity to in SEQ ID NO: 1 in U.S. Pat. No. 6,258,726         or SEQ ID NO: 13 herein.         Paragraph 45. The process of any of paragraphs 1-44, further         wherein a pullulanase is present during liquefaction and/or         saccharification.         Paragraph 46. A composition comprising     -   a bacterial alpha-amylase;     -   a raw starch hydrolyzing alpha-amylase;     -   a carbohydrate-source generating enzyme having a heat stability         at 70° C., pH 5.3, of at least 70%.         Paragraph 47. The composition of paragraph 46, wherein the         alpha-amylase is derived from a strain of the genus Bacillus,         such as a strain of Bacillus stearothermophilus or Geobacillus         stearothermophilus, in particular a variant of a Bacillus         stearothermophilus or Bacillus stearothermophilus alpha-amylase,         such as the one shown in SEQ ID NO: 3 in WO 99/019467 or SEQ ID         NO: 1 herein.         Paragraph 48. The composition of paragraph 46 or 47, wherein the         Bacillus stearothermophilus or Geobacillus stearothermophilus         alpha-amylase is a variant with the following mutations:         I181*+G182*, preferably I181*+G182*+N193F using SEQ ID NO: 3 in         WO 99/019467 or SEQ ID NO: 1 herein for the numbering.         Paragraph 49. The composition of any of paragraphs 46-48,         wherein Bacillus stearothermophilus or Geobacillus         stearothermophilus alpha-amylase is truncated to have around 491         amino acids.         Paragraph 50. The composition of any of paragraphs 46-49,         wherein the bacterial alpha-amylase has at least 80%, more         preferably at least 85%, more preferably at least 90%, more         preferably at least 91%, more preferably at least 92%, even more         preferably at least 93%, most preferably at least 94%, and even         most preferably at least 95%, such as even at least 96%, at         least 97%, at least 98%, at least 99%, or at least 100% identity         to the mature part of the polypeptide of SEQ ID NO: 3 disclosed         in WO 99/019467 or SEQ ID NO: 1 herein.         Paragraph 51. The composition of any of paragraphs 46-50,         wherein the alpha-amylase has a T½ (min) at pH 4.5, 75° C., 0.12         mM CaCl₂) of at least 20, such as at least 25, such as at least         30, such as at least 40, such as at least 50, such as at least         60, such as at least 70, such as at least 80, such as at least         90, such as at least 100, such as at least 110, such as at least         120, such as at least 130, such as at least 140, such as at         least 150, such as at least 160, such as at least 170, such as         at least 180 such as between 20-300, such as between 50-300,         such as between 60-300, such as between 70-300, such as between         80-300, such as between 90-300, such as between 100-300, such as         between 120-300, such as between 140-300 such as between         160-300, such as between 180-300.         Paragraph 52. The composition of any of paragraphs 46-51,         wherein the bacterial alpha-amylase is derived from Bacillus         stearothermophilus alpha-amylase truncated to have around 491         amino acids with the mutations selected from the group         consisting of:     -   V59A+Q89R+E129V+K177L+R179E+I181*+G182*+N193F+H208Y+K220P+N224L+Q254S;     -   E129V+K177L+R179E+I181*+G182*+N193F; and     -   E129V+K177L+R179E+I181*+G182*+N193F+K220P+N224L+S242Q+Q254S.         Paragraph 53. The composition of any of paragraphs 46-52,         wherein the raw starch hydrolyzing alpha-amylase is of fungal         origin, preferably a variant of Rhizomucor pusillus         alpha-amylase with Aspergillus niger glucoamylase linker and SBD         and further one or more of the following substitutions: G128D,         D143N, K192R, such as G128D+D143N or G128D+D143N+K192R (using         SEQ ID NO: 14 herein for the numbering).         Paragraph 54. The composition of any of paragraphs 46-53,         wherein the carbohydrate-source generating enzyme is a         glucoamylase, preferably a glucoamylase having a heat stability         at 70° C., pH 5.3, of at least 75%, preferably at least 80%,         preferably at least 85%.         Paragraph 55. The composition of any of paragraphs 46-54,         wherein the carbohydrate-generating enzyme is a glucoamylase         having a relative activity at pH 4.5 of at least 80%, preferably         at least 85%, preferably at least 90%.         Paragraph 56. The composition of any of paragraphs 46-55,         wherein the carbohydrate-generating enzyme is a glucoamylase         having a pH stability at pH 4.5 of at least at least 80%, at         least 85%, at least 90%, at least 95%, at least 100%.         Paragraph 57. The composition of any of paragraphs 46-56,         wherein the carbohydrate-source generating enzyme is a         glucoamylase, preferably derived from a strain of the genus         Penicillium, especially a strain of Penicillium oxalicum         disclosed as SEQ ID NO: 2 in PCT/CN10/071753 published as WO         2011/127802 or SEQ ID NO: 9 and 15 herein, or a variant thereof         having a K79V substitution (using SEQ ID NO: 15 herein for         numbering).         Paragraph 58. The composition of paragraph 46-57, wherein the         glucoamylase has at least 80%, more preferably at least 85%,         more preferably at least 90%, more preferably at least 91%, more         preferably at least 92%, even more preferably at least 93%, most         preferably at least 94%, and even most preferably at least 95%,         such as even at least 96%, at least 97%, at least 98%, at least         99% or 100% identity to the mature polypeptide shown in SEQ ID         NO: 2 in PCT/CN10/071753 published as WO 2011/127802 or SEQ ID         NO: 9 herein.         Paragraph 59. The composition of any of paragraphs 46-58,         further comprising a protease.         Paragraph 60. The composition of any of paragraphs 46-59,         further comprising a metallo protease.         Paragraph 61. The composition of any of paragraphs 46-60,         wherein the protease has a thermostability of more than 30%,         more than 40%, more than 50%, more than 60%, more than 70%, more         than 80%, more than 90% determined as Relative Activity at 80°         C./70° C.         Paragraph 62. The composition of any of paragraphs 46-61,         wherein the protease has a thermostability of more than 12%,         more than 14%, more than 16%, more than 18%, more than 20%,         determined as Relative Activity at 85° C./70° C.         Paragraph 63. The composition of any of paragraphs 46-62,         wherein the protease is a variant of the metallo protease         derived from Thermoascus aurantiacus CGMCC No. 0670 shown in SEQ         ID NO: 3 herein.         Paragraph 64. The composition of any of paragraphs 46-63,         wherein the protease is derived from a strain of Pyrococcus         Paragraph 65. The composition of any of paragraphs 46-64,         wherein the protease is derived from a strain of Pyrococcus         furiosus.         Paragraph 66. The process of any of paragraphs 46-65 wherein the         protease is the one shown in SEQ ID NO: 1 in U.S. Pat. No.         6,258,726 or SEQ ID NO: 13 herein.         Paragraph 67. The process of any of paragraphs 46-66, wherein         the protease is one having at least 80%, such as at least 85%,         such as at least 90%, such as at least 95%, such as at least         96%, such as at least 97%, such as at least 98%, such as at         least 99% identity to in SEQ ID NO: 1 in U.S. Pat. No. 6,258,726         or SEQ ID NO: 13 herein.         Paragraph 68. The composition of any of paragraphs 46-67,         further comprising a pullulanase.         Paragraph 69. The composition of any of paragraphs 46-68         comprising     -   a bacterial alpha-amylase derived from Bacillus         stearothermophilus;     -   a raw starch hydrolyzing alpha-amylase derived from Rhizomucor         pusillus;     -   a carbohydrate-source generating enzyme having a heat stability         at 70° C., pH 5.3, of at least 70% derived from Penicillium         oxalicum. 

The invention claimed is:
 1. A composition comprising a bacterial alpha-amylase having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1, wherein said bacterial alpha-amylase has alpha-amylase activity; a raw starch hydrolyzing alpha-amylase having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:14 which includes one or more amino acid substitutions selected from the group consisting of: G128D, D143N, K192R, and a combination thereof, wherein said raw starch hydrolyzing alpha-amylase has alpha-amylase activity; and a carbohydrate-source generating enzyme having at least 90% sequence identity to the mature polypeptide of the amino acid sequence of SEQ ID NO 9 which includes a K79V substitution and has a heat stability at 70° C., pH 5.3, of at least 70%, or a carbohydrate-source generating enzyme having at least 90% sequence identity to the mature polypeptide of SEQ ID NO: 15 which includes a K79V substitution and has a heat stability of 70° C., pH 5.3, of at least 70%.
 2. The composition of claim 1, wherein said bacterial alpha-amylase is a Bacillus alpha-amylase.
 3. The composition of claim 1, wherein said bacterial alpha-amylase has at least 95% sequence to the amino acid sequence of SEQ ID NO:
 1. 4. The composition of claim 1, wherein said bacterial alpha-amylase has at least 97% sequence identity to the amino acid sequence of SEQ ID NO:
 1. 5. The composition of claim 3, wherein said bacterial alpha-amylase has at least 99% sequence identity to the amino acid sequence of SEQ ID NO:
 1. 6. The composition of claim 1, wherein said raw starch hydrolyzing alpha-amylase is a variant of Rhizomucor pusillus alpha-amylase having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:14 with a Aspergillus niger glucoamylase linker and a starch binding domain, wherein said raw starch hydrolyzing alpha-amylase comprises one or more of the following substitutions selected from the group consisting of: G128D, D143N, K192R, G128D+D143N, and G128D+D143N+K192R, and wherein said raw starch hydrolyzing alpha-amylase has alpha-amylase activity.
 7. The composition of claim 1, wherein said carbohydrate-source generating enzyme is a glucoamylase having a heat stability at 70° C., pH 5.3, of at least 75%.
 8. The composition of claim 1, wherein said carbohydrate-source generating enzyme is a glucoamylase having a relative activity at pH 4.5 of at least 80%.
 9. The composition claim 1, wherein said carbohydrate-source generating enzyme is a glucoamylase having a pH stability at pH 4.5 of at least 80%.
 10. The composition of claim 1, wherein said carbohydrate-source generating enzyme is a Penicillium glucoamylase.
 11. The composition of claim 10, wherein said glucoamylase is a Penicillium oxalicum glucoamylase comprising the mature polypeptide of the amino acid sequence of SEQ ID NO: 9 and 15, which includes a K79V substitution.
 12. The composition of claim 1, wherein said carbohydrate-source generating enzyme has at least 95% sequence identity to the mature polypeptide of the amino acid sequence of SEQ ID NO:
 9. 13. The composition of claim 1, wherein said carbohydrate-source generating enzyme has at least 97% sequence identity to the mature polypeptide of the amino acid sequence of SEQ ID NO:
 9. 14. The composition of claim 1, wherein said carbohydrate-source generating enzyme has at least 99% sequence identity to the mature polypeptide of the amino acid sequence of SEQ ID NO:
 9. 15. The composition of claim 1, further comprising a protease.
 16. The composition of claim 1, further comprising a pullulanase.
 17. The composition of claim 1, further comprising a protease and a pullulanase. 